Compositions and methods for inhibiting nf-kappab mediated tumorigenicity and adhesion dependent survival of cancer cells

ABSTRACT

Disclosed are compositions and methods for inhibiting NF-κB mediated cellular proliferation and metastasis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority upon U.S. application Ser. No.60/358,853, filed on Feb. 21, 2002, which is herein incorporated byreference in its entirety.

I. BACKGROUND

Many adherent cells undergo apoptosis when converted to suspensionculture or when detached from the underlying extracellular matrix.(Strater et al., 1996) This process has been termed anoikis and may bean important mechanism of tissue homeostasis. (Frisch and Francis, 1994)Cancer cells, on the other hand, have long been known for their abilityto grow in the absence of adhesion, a characteristic known asanchorage-independent proliferation. (Martin, 1996) This is of clinicalrelevance to metastasis, because cancer cells must transiently survivein the absence of adhesion as they travel to and migrate into distanttissues via the circulatory or lymphatic systems. Efforts to curbmetastasis are critical since the presence of metastases is the singlemost important prognostic indicator for survival in cancer patients.Preventing metastasis of primary tumors is hampered by the apparent easeby which cancer cells gain access to the circulatory system eithernaturally or at the time of surgical resection. (Hansen et al., 1999;Hansen et al., 1995; Mehes et al., 2001) latrogenic seeding of cancercells is particularly worrisome as the primary modality of therapy formost resectable solid tumors is surgery.

Disclosed are compositions and methods related to inhibiting the affectof NF-κB on cancer, through inhibition of metastasis, readhesion, andcancer cell proliferation.

II. SUMMARY

The disclosed compositions and methods are related to the treatment andinhibition of cancer.

III. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of NF-κB inhibitors on colon cancer cellproliferation. DLD-1, HCT-116, and HT-29 were cultured on 96-well platesin the presence or absence of BAY 11-7082 (A) or BAY 11-7085 (B) for 8days. The cells were fixed, stained with crystal violet, solubilized indeoxycholate, and read in a spectrophotometer at 590 nm. Legend: control(DMSO) squares, BAY 0.1 μM diamonds, BAY 1.0 μM triangles, BAY 10.0 μMovals. The data points represent the mean values of experimentsperformed in triplicate and the error bars the standard error of themeans. FIG. 1C shows electrophoretic mobility shift assays showinginhibition of NF-κB binding to an NF-κB DNA consensus oligonucleotide innuclear lysates from HT-29 cells treated with BAY 11-7082 or BAY 11-7085for 3 (left panel) or 6 (right panel) hours. The figure shows thetypical results of four experiments. The densitometry data of the gelsare shown in graphic form at the bottom of the figure. Excess coldcompetitor DNA oligonucleotide was used to demonstrate the specificityof the protein-DNA binding.

FIG. 2A shows NF-κB inhibitors, BAY 11-7082 and BAY 11-7085, decreaseanchorage-independent proliferation of colon cancer cells. DLD-1,HCT-116, and HT-29 cells were cultured for 6 days in soft agar in thepresence of DMSO (control), 10 μM BAY 11-7082, or 10 μM BAY 11-7085. Thedata points represent the means of colony counts per field (20×)performed in 5 different regions of the culture dishes. The data shownrepresent a typical result of two separate experiments. FIGS. 2B,C, showathymic mice received subcutaneous injections of HT-29 (B) or HCT-116(C) cells, followed by 5 mg/ml of BAY 11-7085 (dark bars) or DMSO (whitebars) intraperitoneally twice weekly. The bars represent the 25-75%ranges of the tumor volumes and the horizontal lines in the bars are themedians. Using the Kruskal-Wallace statistical test to compare themedian tumor volumes between BAY 11-7085 and DMSO yielded a p-value of0.005 for HT-29 cells but >0.05 for HCT-116 cells.

FIG. 3 shows NF-κB inhibitors cause apoptosis of colon cancer cells.FIG. 3A shows adherent and HT-29 cells were treated with DMSO alone(control), 10 and 20 μM BAY 11-7085, or 20 μM MG-132 for 24 hours. Thedata points represent the average percent of annexin V-positive,propidium iodide-negative cells (of the total cells counted) fromtriplicate experiments. The error bars represent the standard error ofthe means. FIG. 3B shows adherent HT-29 cells were treated with BAY11-7085 at various concentrations for 24 hours after which thenonadherent and adherent cells were collected separately. The resultsshown are a graphic representation of individual flow cytometry resultsexpressed as the percentage of cells that were annexin V-positive ofthose that excluded propidium iodide. FIG. 3C shows adherent HCT-116cells were treated with 20 μM MG-132 for 24 hours after which the cellswere collected. An immunoblot of the lysates for cleaved PARP wasperformed. Each lane represents equal total protein concentrations. FIG.3D shows confluent monolayers of HT-29 cells were treated with variousconcentrations of BAY 11-7085 for 3 hours after which they weredispersed with trypsin-PBS. The cells were allowed to adhere to plasticdishes for 1 hour after which they were washed gently in PBS and stainedwith crystal violet. The crystal violet stained cells were solubilizedin deoxycholate and the absorbance detected in a spectrophotometer at590 nm. The adherent cells were expressed as a fraction of the controlsEach data point represents the average of triplicate experiments and theerror bars the standard errors of the means.

FIG. 4A shows transient suspension of colon cancer cells greatlyincreases their susceptibility to apoptosis in the presence of BAY11-7085. HT-29 cells that were either transiently suspended withtrypsin-EDTA or adherent for three days, were treated at time zero withDMSO or 10-100 μM BAY 11-7085. The percent of apoptotic cells wasdetermined using the annexin V flow cytometry assay and graphed versusthe dose of BAY 11-7085. FIG. 4B shows DLD-1, FIG. 4C shows HCT-116, andFIG. 4D shows HT-29 cells that were transiently suspended intrypsin-EDTA and allowed to readhere to glass coverslips for 1-24 hours.The cells were fixed and immunostained with an NF-κB p65 subunitmonoclonal antibody. Note the strong nuclear staining at 1 hour versusthe markedly reduced nuclear staining of the cells.

FIG. 5A shows NF-κB binding activity assay (TransAM) of DLD-1, HCT-116,and HT-29 cells that were transiently suspended by scraping or withtrypsin-EDTA. The transiently suspended cells were lysed and allowed tobind to DNA oligonucleotides, containing the NF-κB consensus bindingsite, that were immobilized to 96-well plates. NF-κB protein bound tothe oligonucleotides was detected by an antibody to the p65 subunit thatonly recognized p65 that is bound to DNA. The positive control was HeLacells that were stimulated with TNFα A control was performed usingexcess free NF-κB oligonucleotides (Competitor). FIG. 5B shows HT-29cells were transiently suspended and allowed to readhere in the presenceor absence of BAY 11-7085 for three hours after which they were lysed.NF-κB binding activity was determined by the TransAM assay as in FIG.5A. FIG. 5C shows transiently suspended DLD-1, HCT-116, and HT-29 cellswere allowed to readhere in the presence of DMSO or BAY 11-7085 for 8hours after which the percent of apoptotic cells was determined by theflow cytometric annexin V assay. The data shown represent typicalresults of three separate experiments. FIG. 5D shows 293-COX-2 cellswere incubated in the presence or absence of 1 μg/ml of ponasterone for48 hours after which they were lysed. An immunoblot for COX-2 wasperformed. FIG. 5E shows 293-COX-2 cells were incubated in the presenceor absence of ponasterone for 48 hours followed by 8 hours of exposureto DMSO or BAY 11-7085. Note that induction of COX-2 resulted in anincreased susceptibility to BAY 11-7085-induced apoptosis.

FIG. 6 shows an intraabdominal seeding model. FIGS. 6A, B, C show tumorimplants of the liver of three athymic mice 21 days after receivingintraperitoneal injections of HT-29 cells. The mice were pretreated andtreated for a total of 21 days with DMSO twice weekly. FIG. 6D showsintestinal tumor implants (arrows) of an athymic mouse 21 days afterintraperitoneal injections of HT-29 cells. The mouse was pretreated andtreated for a total of 21 days with DMSO twice weekly. FIG. 6E shows amicrograph showing tumor invasion through the liver capsule. Thissection was obtained from the tumor implant from the mouse in FIG. 6A.FIG. 6F shows a peritoneal tumor implant taken from the bowel wall ofthe mouse in FIG. 6A. FIG. 6G, (liver), FIG. 6H, (abdominal wall), andFIG. 6I, (intestinal) show tumor implants in three athymic mice 21 daysafter receiving intraabdominal injections of HCT-116 cells. All of themice were treated with 5 mg/kg of BAY 11-7085 twice weekly.

FIG. 7A shows DLD-1 HCT-116, and HT-29 cells were cultured in 96-wellplates for 10 days in the presence or absence of 10 or 50 μg/ml of cA2.At various time-points the cells were the cells were fixed, stained withcrystal violet, solubilized in deoxycholate, and read in aspectrophotometer at 590 nm. The data points represent the means oftriplicate experiments and the error bars the standard errors of themeans. FIG. 7B shows adherent HT-29 cells were pretreated with 50 μg/mlof cA2 for 48 hours followed by the addition of DMSO or 20 μM BAY11-7085 for another 24 hours. The percent of apoptotic cells was thendetermined by the annexin V assay. Similar results were obtained forDLD-1 cells. The results shown are typical of two separate experiments.FIG. 7C shows adherent or transiently suspended HT-29 cells were treatedwith 20 μM BAY 11-7085 for 8 hours after which they were lysed.Immunoblots were performed for c-IAP-2, TRAF-1, TRAF-2, and FLIP. Alllanes contain equal total protein. FIG. 7D shows a FLIP immunoblot ofDLD-1, HCT-116, and HT-29 lysates.

FIG. 8 shows HT-29 cells that were transiently suspended and allowed toreadhere in the presence or absence of various concentrations of theNF_(κ)B inhibitors, PDTC, BAY 11-7085, and MG-132 for 4 h. Thepercentage of apoptotic cells was determined using the annexin V assay.The data points represent the averages of three experiments; bars, ±SE.E. DLD-1 and F. HT-29 cells were transduced with an adenoviruscontaining the I_(κ)B super-repressor construct. After 3 days, the cellswere transiently suspended and allowed to readhere in the presence ofsubapoptotic concentrations of BAY 11-7085 or DMSO (vehicle) for 8 h.The data are expressed as fractions of cells surviving compared with thecontrols (no adenovirus) and were quantitatively determined by stainingthe remaining adherent cells with crystal violet.

IV. DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that thedisclosed compositions and methods are not limited to specific syntheticmethods, specific recombinant biotechnology methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

A. DEFINITIONS

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” as well as “less than” and “greater than” 10 are alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point 15 are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Variables such as R₁—R₇, X, and Y used throughout the application arethe same variables as previously defined unless stated to the contrary.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and thelike.

The term “alkenyl” as used herein refers to a hydrocarbon group of 2 to24 carbon atoms and structural formula containing a carbon-carbon doublebond.

The term “alkynyl” as used herein refers to a hydrocarbon group of 2 to24 carbon atoms and a structural formula containing a carbon-carbontriple bond.

The term “halogenated alkyl” as used herein refers to an alkyl group asdefined above with one or more hydrogen atoms present on the alkylgroups substituted with a halogen (F, Cl, Br, I).

The term “aromatic” is defined as any carbon-based aromatic groupincluding, but not limited to, benzene, naphthalene, etc. The term“aromatic” also includes “heteroaromatic,” which is defined as anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group.

The term “substituted aromatic” is defined as an aromatic group havingat least one group attached to the aromatic group that is not hydrogen.Examples of groups that can be attached to the aromatic group include,but are not limited to, alkyl, alkynyl, alkenyl, aryl, heterocyclic,halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid,or alkoxy.

The term “aralkyl” is defined as an aryl group having an alkyl, alkynyl,or alkenyl group attached to the aromatic group. An example of anaralkyl group is a benzyl group.

B. COMPOSITIONS AND METHODS 1. NF-κB

NF-κB is a heterodimeric transcription factor comprised of variousprotein subunits: p50/p105, p65/RelA, p52/p100, c-Rel, and RelB. NF-κBis localized to the cytoplasm by association with the IκB proteins,which inhibit translocation of NF-κB into the nucleus thus preventingits transcriptional activity. (Beg et al., 1992) Under the influence ofcytokines, reactive oxygen species, growth factors, and other stimuli,IκB kinases (IKK) phosphorylate IκB proteins on two critical serineresidues. (DiDonato et al., 1996; Traenckner et al., 1995) This thentargets the IκB proteins for ubiquitination and subsequent degradationby the proteasome, which results in free NF-κB that translocates intothe nucleus and activates the transcription of various genes possessingκB consensus DNA binding sites in their promoters. (Alkalay et al.,1995; Chen et al., 1995; Henkel et al., 1993). Many of the genesregulated by NF-κB encode proteins that promote inflammation, includingCOX-2, (Crofford et al., 1997; Newton et al., 1997a; Newton et al.,1997b; Schmedtje et al., 1997) and angiogenesis factors such asVEGF.(Huang et al., 2001). In addition, NF-κB mediates the transcriptionof several survival genes, c-Myc, Bcl2, p53, p21, c-FLIP, c-IAP-1,c-IAP-2, XIAP, IEX-1L COX-2, TRAF-1, and TRAF-2. (Kreuz et al., 2001;Schwartz et al., 1999; Stehlik et al., 1998; Wang et al., 1998; Wu etal., 1998).

Disclosed herein soluble NF-κB inhibitors inhibit anchorage-dependentand -independent proliferation, and tumorigenicity, and cause apoptosisof cancer cells including colon cancer cells. The induction of apoptosisof colon cancer cells by NF-κB inhibition occurs in a celladhesion-dependent fashion. Readhesion following transient suspension ofthe cell lines causes a large activation of NF-κB, which rendered thecells exquisitely sensitive to NF-κB inhibitor-induced apoptosis.Furthermore, pretreatment of athymic mice with an NF-κB inhibitorcompletely prevented liver metastasis following intraperitoneal deliveryof a colon cancer cell line. Disclosed herein colon cancer cells, aswell as cancer cells related to colon cancer cells, utilize NF-κB formitogenesis and as a major survival factor during readhesion.

2. NF-κB Inhibitors

The NF-κB inhibitors disclosed herein can be any molecule that inhibitsNF-κB function. It is understood that NF-κB function can be inhibited oraltered in many ways. For example, NF-κB function can be inhibited bythe NF-κB inhibitor directly interacting with NF-κB. Directlyinteracting with NF-κB means that the inhibitor touches or binds withNF-κB. The NF-κB inhibitor can also indirectly inhibit NF-κB function.Indirectly inhibiting the function of NF-κB means that the NF-κBinhibitor does not touch or bind NF-κB. A molecule would indirectlyinhibit NF-κB function, by for example, reducing the expression oractivation or nuclear transport of NF-κB.

One example of indirect NF-κB inhibitors are NF-κB inhibitors thatinhibit NF-κB transport into the nucleus. NF-κB remains cytoplasmic ifit interacts with IκB. When IκB is phosphorylated, it causes IκB to beubiquinated, and degraded, which allows NF-κB to transport to thenucleus. Disclosed are compositions which inhibit NF-κB transport to thenucleus through interactions with IκB which prevent IκB from interactingwith NF-κB. One example of indirect NF-κB inhibitors that inhibit NF-κBtransport into the nucleus are NF-κB inhibitors that inhibit IκBphosphorylation. Another example of indirect NF-κB inhibitors are NF-κBinhibitors that inhibit expression of NF-κB. Another example of indirectNF-κB inhibitors are NF-κB inhibitors that inhibit translation of NF-κB.

Typically the disclosed NF-κB inhibitors inhibit TNFα induced NF-κBactivation. TNFα typically causes an activation of NF-κB. The disclosedinhibitors decrease this TNFα-induced activation. Many assays can beused to determine if NF-κB inhibitors are decreasing TNFα-dependentactivation.

Disclosed are NF-κB inhibitors that can inhibit anchorage dependent andindependent proliferation and tumorigenicity and can cause apoptosis.Also disclosed are NF-κB inhibitors that inhibit induction of apoptosisby NF-κB inhibition which occurs in a cell adhesion dependent fashion.Disclosed are NF-κB inhibitors that affect cells where readhesion ofcells causes a large activation of NF-κB which causes the cells tobecome sensitized to NF-κB inhibitor induced apoptosis.

Disclosed herein are colon cancer cells, as well as other cancer cells,which utilize NF-κB for mitogenesis and are a survival factor duringreadhesion. Disclosed is that NF-κB inhibitors diminish colon cancercell proliferation.

BAY 11-7082 and BAY 11-7085 inhibit IκB phosphorylation and TNFα inducedNF-κB activation. Disclosed herein, BAY 11-7082 inhibits growth of DLD-1and HCT-116 cancer cell lines.

Disclosed herein NF-κB inhibitors inhibit colon cancer celltumorigenicity.

Disclosed herein, cells that contain APC mutations are susceptible toBAY 11-7085 and related molecules. For example, cells obtained from celllines DLD-2 and HT-29 cells are susceptible to BAY 11-7085. DLD-1 andHT-29 contain APC mutations. HCT-116 cells have activating mutations onB-catenin which is normally regulated by the APC product. Also HCT-116cells do not express COX-2. COX-2 is commonly over-expressed incolorectal cancers, but HCT-116 cells do not.

Disclosed herein anchorage independent inhibition of proliferation isinhibited by both BAY11-7082 and 7085 in DLD-1 and HT-29 cells, and bothBAY 11-/082 and BAY 11-7085 reduce tumor volumes in vivo in athymic miceinjected with HT-29 tumor cells.

Disclosed herein MC-132 and PDTC cause an increase in apoptosis inadhered cells and BAY-11-7085 and BAY-11-7082 cause apoptosis in cellseven when not adhered.

C. METHODS OF USING THE COMPOSITIONS

The disclosed compositions can be used in a variety of ways as researchtools. For example, the disclosed compositions, such as BAY-11-7085 andBAY-11-7082, PDTC and MC-132 can be used as reagents and standards incellular proliferation assays and as NF-κB inhibitors for assays relatedto cancer.

The compositions can be used for example as competitive inhibitors incombinatorial chemistry protocols or other screening protocols toisolate molecules that possess desired functional properties related toinhibition of cancer or metastasis of cell systems disclosed herein.

1. Method of Treating Cancer

Disclosed herein NF-κB inhibitors can be given to a subject. Any subjectin need of the NF-κB inhibitors as disclosed herein can be given theNF-κB inhibitors. The subject can, for example, be a mammal, such as amouse, rat, rabbit hamster, dog, cat, pig, cow, sheep, goat, horse, orprimate, such as monkey, gorilla, orangutan, chimpanzee, or human.

Disclosed herein NF-κB inhibitors can be used for inhibiting cancer cellproliferation. Inhibiting cancer cell proliferation means reducing orpreventing cancer cell growth. Inhibitors can be determined by using acancer cell assay. For example, either a DLD-1, HCT-116, and HT-29 cellline can be cultured on 96-well plates in the presence or absence of theinhibitor for 8 days. The cells can be fixed, stained with crystalviolet, solubilized in deoxycholate, and read in a spectrophotometer at590 nm. In certain embodiments the inhibitors are those that willinhibit 10% or 15% or 20% or 25% or 30% or 35% or 40% or 45% or 50% or55% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% of the cellgrowth relative to a control as determined by spectrophotometry.

In certain embodiments the inhibitors also inhibit anchorage-dependentproliferation of cancer cells, such as colon cancer cells. Inhibitorscan be assayed using a soft agar colony formation assay. For example,DLD-1, HCT-116, and HT-29 cells can be cultured for 6 days in soft agarin the presence of DMSO (control) or inhibitor. The number of colonycounts can then be compared by, for example, taking a percentage of thecells formed relative to a control. In certain embodiments theinhibitors are those that will inhibit 10% or 15% or 20% or 25% or 30%or 35% or 40% or 45% or 50% or 55% or 60% or 65% or 70% or 75% or 80% or85% or 90% or 95% of the cell growth relative to a control as determinedby counting colonies formed.

Disclosed herein NF-κB inhibitors can be used for promoting cancer cellapoptosis. Promoting cancer cell apoptosis means causing the cell todie. An apoptosis assay can be used to determine if the inhibitorspromote cancer cell apoptosis. For example, an apoptosis assay asdisclosed in Example 1 can be used. In certain embodiments, the percentof apoptosis was determined as the percent of annexin V-positive,propidium iodide-negative cells of the total cells counted in anApoptosis assay, such as that disclosed in Example 1. In certainembodiments, the inhibitor causes at least 10% of the cells to beapoptotic or the inhibitor causes at least 15% of the cells to beapoptotic or the inhibitor causes at least 20% of the cells to beapoptotic or the inhibitor causes at least 25% of the cells to beapoptotic or the inhibitor causes at least 30% of the cells to beapoptotic or the inhibitor causes at least 35% of the cells to beapoptotic or the inhibitor causes at least 40% of the cells to beapoptotic or the inhibitor causes at least 45% of the cells to beapoptotic or the inhibitor causes at least 50% of the cells to beapoptotic or the inhibitor causes at least 55% of the cells to beapoptotic or the inhibitor causes at least 60% of the cells to beapoptotic or the inhibitor causes at least 65% of the cells to beapoptotic or the inhibitor causes at least 70% of the cells to beapoptotic or the inhibitor causes at least 75% of the cells to beapoptotic or the inhibitor causes at least 80% of the cells to beapoptotic or the inhibitor causes at least 85% of the cells to beapoptotic or the inhibitor causes at least 90% of the cells to beapoptotic or the inhibitor causes at least 95% of the cells to beapoptotic.

In certain embodiments, the apoptotic effect of the disclosed inhibitorsis enhanced in cells that are transiently suspended, such as metastaticcells circulating through the blood stream, prior to readhesion.Inhibitors that have this property can be determined by assaying theirapoptotic effect as, for example described herein, in a transientsuspension assay, as for example described in Examples 1 and 2. Forexample, cells can be put into suspension by scraping and then allowedto readhere. Inhibitors that cause an increase in apoptosis uponreadhering to the substrate are disclosed. An increase upon readheringto the substrate can be determined by, for example, looking at multiplesof cells that were apoptotic upon readhering to the substrate comparedwith cells that were apoptotic upon remaining in suspension. Disclosedare inhibitors wherein the inhibitor causes at least 1.5 fold or 2 foldor 3 fold or 4 fold or 5 fold or 6 fold or 7 fold or 10 fold or 15 foldor 20 fold or 30 fold or 50 fold or 100 fold more apoptosis of cellsthat readhere then apoptosis of cells that remain in suspension. (seefor example FIG. 3).

In certain embodiments, the inhibitor causes at least 17% of the cellsto be apoptotic.

Disclosed herein NF-κB inhibitors can be used for inhibiting readhesionof cancer cells to a surface. Inhibiting readhesion of cancer cells to asurface means decreasing the number of cells capable of readhering to asurface after being transiently suspended as discussed herein.

Disclosed herein NF-κB inhibitors can be used for inhibiting metastasisof cancer cells. Inhibiting metastasis of cancer cells means decreasingor lowering the amount of metastatic tumors that arise in an organism.For example, disclosed are inhibitors that inhibit metastasis in an invivo assay. One way of performing an in vivo assay to determine if aninhibitor inhibits metastasis is to inject a cancer cell line, such asHT-29, into the abdominal cavity of an athymic mouse. Mice arepretreated with the inhibitor or a control intraperotneally, forexample. The mouse can then be treated regularly, for example, twiceweekly with vehicle or BAY 11-7085 for a period of time, for example, 21days. The mouse can then be sacrificed and assayed for metastatic tumorformation. Disclosed are compositions which inhibit metastatic tumorformation in this type of assay disclosed herein, as well ascompositions that reduce metastatic tumor formation by at least 10% or15% or 20% or 25% or 30% or 35% or 40% or 45% or 50% or 55% or 60% or65% or 70% or 75% or 80% or 85% or 90% or 95% relative to a controlcompound.

For example, NF-κB inhibitors can inhibit intraabdominal metastasisarising from, for example, colon or rectal cancers. NF-κB inhibitors canalso inhibit hepatic, parietal or peritoneal metastasis arising from,for example, colon or rectal cancers.

Disclosed herein NF-κB inhibitors can be used for inhibitingtumorigenesis. Inhibiting tumorigenesis means decreasing or lowering theamount of tumors present in an organism. For example, disclosed areinhibitors that inhibit tumorigenesis in an in vivo assay. One way ofperforming an in vivo assay to determine if an inhibitor inhibitstumorigenesis is to inject a cancer cell line subcutaneously, such asHT-29, into an athymic mouse, such as a female mouse. The mouse can thenbe treated regularly, for example, twice weekly with vehicle or BAY11-7085 for a period of time, for example, 21 days or 28 days. The mousecan then be sacrificed and assayed for tumor formation and size.Disclosed are compositions which inhibit tumorigenesis in this type ofassay disclosed herein, as well as compositions that reducetumorigenesis by at least 10% or 15% or 20% or 25% or 30% or 35% or 40%or 45% or 50% or 55% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or95% relative to a control compound.

Disclosed herein NF-κB inhibitors can be administered to cells thatutilize NF-κB for mitogenesis. Disclosed herein NF-κB inhibitors can beadministered to cells that utilize NF-κB for readhesion. A cell can bedetermined to utilize NF-κB for readhesion if in a transient suspensionassay, upon readhesion NF-κB is activated as determined by any assaythat looks at NF-κB activation. For example, NF-κB binding assays to theconsensus sequence can be used, such as Trans-AM® assay the discussed inExamples 1 and 2. If the NF-κB activation is greater upon readhesionthen in suspension, the cells can be considered to utilize NF-κB forreadhesion.

Disclosed herein NF-κB inhibitors can be administered to cells to inducethe apoptosis of the cells in a TNFα independent manner. TNFα canactivate apoptosis, but the NF-κB inhibitors can be administered tocause apoptosis in a TNFα independent manner. The disclosed inhibitorscan promote apoptosis of cancer cells, such as colon cancer cells,without TNFα. An inhibitor or cell can be shown to be apoptotic withoutneeding the inhibitor by using TNFα inhibiting antibody, such as cA2,and seeing that apoptosis is still caused by the inhibitor even in thepresence of the TNFα inhibiting antibody, such as cA2.

When the NF-κB inhibitors are administered, they typically cause adecrease in the expression of anti-apoptotic proteins. Expression of theanti-apoptotic proteins can be determined by any means for determiningexpression. For example, standard biotechnology methods such as PCR orNorthern blots can be used to determine the expression levels ofanti-apoptotic genes. A decrease can be determined by assaying theexpression levels of a desired anti-apoptotic gene in the presence of apotential inhibitor and comparing this level of expression to the levelof expression in the absence of the inhibitor. Disclosed are inhibitors,which decrease the expression of the anti-apoptotic genes in such anassay.

As discussed herein NF-κB inhibitors can, for example, be used to reducethe proliferation of cancer cells, as well as to cause the apoptosis ofcancer cells or inhibit the readhesion of cancer cells or inhibit themetastasis of cancer cells. NF-κB inhibitors can, for example, beadministered to any cancer cell that uses NF-κB to survive or metastizeor adhere or which activates NF-κB during its life cycle.

NF-κB inhibitors can be administered to cancer cells that have amutation in the adenomatous polyposis coli (APC) gene. APC has beenshown to be a tumor suppressor gene in, for example, colon cells (seefor example, Groden et al., Cancer Research, 55 1531-1539 (1995) hereinincorporated by reference at least for material related to APC mutationsand assays of the same). A variety of different morphologies of theeffect of the mutated APC can be seen. Whether a cell contains an APCmutation can be assayed for using standard recombinant biotechnologyprotocols, for example, sequencing and PCR analysis or ligation mediatedchain reaction (LCR) or other methods using (for example chiptechnology) capable of assaying and comparing DNA sequences. Themutations can readily be assayed as being functional mutations, by forexample, expressing the mutant protein in a cell and determining if themutant protein induces an oncogenic phenotype. Assays for determiningthe effect of an APC mutation can also be performed as discussed inGroden et al. In certain embodiments an activating mutation can bedetermined by assaying the mutation and comparing the effects to theeffects of APC mutations in DLD-1 cells or HT29 cells. For example, asin Groden a fully functional APC gene can be transfected into a DLD-1 orHT29 cell, and this decreases the oncogenic behavior of the DLD-1 orHT29 cells because the non-mutant APC gene rescues normal phenotype. Incertain embodiments, a given APC mutation can be assayed by, forexample, transfecting the mutant APC into either a DLD-1 or HT29 cellline and comparing the level of rescue provided by the mutant APC to thelevel of rescue of the non-mutant APC. In certain embodiments the APCmutant will be considered an in-activating APC mutant, i.e. a mutantwhich causes oncogenic phenotype, if the rescue of the cells transfectedby the mutant APC is less than 10% or 15% or 20% or 25% or 30% or 35% or40% or 45% or 50% or 55% or 60% or 65% or 70% or 75% or 80% or 85% or90% or 95% of the rescue of cells transfected with the non-mutant APC,as judged by any of the criteria used to judge oncogenic phenotype ofDLD-1 or HT29 cells.

In certain embodiments, NF-κB inhibitors are not administered to cancercells that have an activating mutation on β-catenin. Whether a cellcontains a β-catenin mutation can be assayed for using standardrecombinant biotechnology protocols, for example, sequencing and PCRanalysis. The mutations can be assayed for function as discussed herein.

Disclosed herein NF-κB inhibitors can be administered to cancer cellsthat express the COX-2 gene. A cell expresses the COX-2 gene if thereare detectable transcripts of the COX-2 gene in the cell using an assayto detect transcripts, such as a hybridization assay, such as a northernblot or any of the chip type assays available, or an amplification basedassay based on, for example, PCR or other amplification methods. Incertain embodiments, the NF-κB inhibitors can be administered to cancercells that over express the COX-2 gene. Over expression of COX-2 can bedetermined by using any of the methods and assays discussed for theexpression of COX-2 and comparing the level of expression to that of acontrol population of cells. In general cells do not show activatedCOX-2 expression, even if there is a basal amount of COX-2 expression,but upon additions of mitogens, for example, COX-2 expression, relativeto expression in the absence of mitogens, increases. Thus, a give cancercell or cancer cell line can be assayed for COX-2 expression and thiscan be compared to the COX-2 expression of this cell type in the absenceof the oncogenic phenotype. In certain embodiments a given cancer cellline can be considered to over express COX-2 if the expression is atleast 10% or 15% or 20% or 25% or 30% or 35% or 40% or 45% or 50% or 55%or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% of theexpression of COX-2 in DLD-1 cells or HT29 cells, when expression ofCOX-2 of the cell of interest and DLD-1 or HT29 are assayed in parallel.

Disclosed herein NF-κB inhibitors can be administered to cancer cellsthat express the COX-2 gene as well as having mutations in the APC gene.

It is understood that certain cancers can give rise to cancer celllines. Typically a cancer cell line are cells that are maintained incell culture, but that arose from a specific type of cancer. NF-κBinhibitors can be used for a variety of cancers, but can, for example,be used for cancers that are related to the DLD-1 cancer cell line andthe HT-29 cancer cell line. The DLD-1 cancer cell line and the HT-29cancer cell line, arose from colon cancer cells. Also disclosed arecancer cell lines having the properties of the DLD-1 cancer cell lineand the HT29 cell line.

The disclosed compositions can be used to treat any disease whereuncontrolled cellular proliferation occurs such as cancers. Anon-limiting list of different types of cancers is as follows: lymphomas(Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomas of solidtissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas,high grade gliomas, blastomas, neuroblastomas, plasmacytomas,histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas,AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers ingeneral.

A representative but non-limiting list of cancers that the disclosedcompositions can be used to treat is the following: lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, kidney cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,prostate cancer, skin cancer, liver cancer, melanoma, squamous cellcarcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, and epithelialcancer, renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, stomach cancer, head and neck carcinoma, large bowel cancer,hematopoietic cancers; testicular cancer, colon and rectal cancers,prostatic cancer, or pancreatic cancer.

Compounds disclosed herein may also be used for the treatment ofprecancer conditions such as cervical and anal dysplasias, otherdysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, andneoplasias.

As disclosed herein, the NF-κB inhibitors prevent readhesion oftransiently suspended cancer cells, for example, colon cancer cells, andfor example, cancer cells related to HT29 and DLD1 cell lines. Also, itis well understood that during surgery to remove solid tumors, asdiscussed herein, cancer cells can break of and become transientlysuspended in the subject's circulatory system, only to potentiallyreadhere at a new spot, to seed a new tumor growth. This reseedingoccurs during metastasis as well, but can be accelerated during surgicalresection of the tumor. Thus, the disclosed NF-κB inhibitors can be usedto prevent or inhibit the potential reseeding that can occur during orafter surgical resection of a tumor. By administering the NF-κBinhibitors prior to or after the resection, the transiently suspendedcells can be efficiently caused to apoptos with the NF-κB inhibitors.

Thus disclosed are methods of inhibiting cancer cell proliferation in asubject comprising administering an NF-κB inhibitor to the subject,wherein the subject has had a tumor resected.

Also disclosed are methods, wherein the NF-κB inhibitor is administeredprior to the resection or after the resection or both.

Disclosed are methods, wherein the NF-κB inhibitor is administeredwithin 10 days or 5 days or 1 day or 10 hours or 5 hours or 1 hour or0.5 hours, of the resection.

D. COMPOSITIONS

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular composition such as BAY 11-7082 is disclosedand discussed and a number of modifications that can be made to a numberof molecules including BAY 11-7082 are discussed, specificallycontemplated is each and every combination and permutation of BAY11-7082 and the modifications that are possible unless specificallyindicated to the contrary. Thus, if a class of molecules A, B, and C aredisclosed as well as a class of molecules D, E, and F and an example ofa combination molecule, A-D is disclosed, then even if each is notindividually recited each is individually and collectively contemplatedmeaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F areconsidered disclosed. Likewise, any subset or combination of these isalso disclosed. Thus, for example, the sub-group of A-E, B-F, and C-Ewould be considered disclosed. This concept applies to all aspects ofthis application including, but not limited to, steps in methods ofmaking and using the disclosed compositions. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the disclosed methods.

1. NF-κB Inhibitors

The disclosed method involve NF-κB inhibitors. An NF-κB inhibitor can beany composition that causes a decrease in the expression ofanti-apoptotic proteins. The NF-κB inhibitor can also be any compositionwherein the composition inhibits IκB phosphorylation. The NF-κBinhibitor can also be any composition, wherein the composition inhibitsTNFα induced NF-κB activation.

In certain embodiments, the inhibitors useful in any of the methods areolefins. An “olefin” is defined herein as any compound or moleculepossessing at least one carbon-carbon double bond. Each carbon atom ofthe carbon-carbon double bond may be unsubstituted or independentlysubstituted with one or two different moieties.

In certain embodiments, the inhibitor is an olefin having at least oneelectron-withdrawing group. In another embodiment, the inhibitor is anolefin having at least two electron-withdrawing groups. In this case,when two electron-withdrawing groups are present, theelectron-withdrawing groups can be present on the same olefinic carbonatom or one electron-withdrawing group can be on each olefinic carbonatom.

The term “electron-withdrawing group” is any group that has an affinityor attraction for electron density. For example, when anelectron-withdrawing group is attached to an olefinic carbon atom(C_(α)), then the other olefinic carbon atom (C_(β)) is more susceptibleto nucleophilic attack (i.e., C_(β) is more electropositive) whencompared to an olefin that does not possess an electron-withdrawinggroup. Generally, electron withdrawing groups possess one or morecarbon-carbon multiple bonds, carbon-heteroatom multiple bonds, orheteroatom-heteroatom multiple bonds. Examples of electron-withdrawinggroups include, but are not limited to, a cyano group, a sulfo-oxygroup, a phospho-oxy group, a carboxyl group, a nitro group, a halogen,a halogenated alkyl group, an unsubstituted aromatic ring, or asubstituted aromatic ring having at least one cyano group, sulfo-oxygroup, phospho-oxy group, carboxyl group, hydroxyl group, amino group,ether group, halogenated alkyl group, halogen, or nitro group.

The term “phospho-oxy group” is a group having one of the followingstructures

wherein R¹ is hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl,aralkyl, or substituted or unsubstituted aromatic.

The term “sulfo-oxy group” is a group having one of the followingstructures

wherein R² is hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl,aralkyl, or substituted or unsubstituted aromatic. In one embodiment,the inhibitor is an olefin having a cyano group and a sulfo-oxy grouphaving the structure

wherein R² is hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl,aralkyl, or substituted or unsubstituted aromatic.

In another embodiment, the inhibitor has the structure I.

wherein R³, R⁴ and R⁵ are, independently, hydrogen, alkyl, halogenatedalkyl, alkenyl, alkynyl, aralkyl, or substituted or unsubstitutedaromatic, wherein the compound is the E- or Z-isomer. Thestereochemistry about the carbon-carbon double bond will vary dependingupon the relative positions of the cyano group (—CN) and the sulfonylgroup (—S(O₂)R⁵)). When the cyano group and sulfonyl group are, cis toone another, then the compound is the Z-isomer, and when the cyano groupand sulfonyl group are trans to one another, then the compound is theE-isomer. In one embodiment, the inhibitor having the structure I is theE-isomer.

In one embodiment, when the inhibitor has the structure I, R³ and R⁴ arehydrogen. In another embodiment, R⁵ is methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tertiary butyl, substituted or unsubstituted phenyl, orbenzyl. In a further embodiment, R⁵ is a phenyl group having at leastone alkyl group.

In another embodiment, the inhibitor having the structure I is the ethylester of2-cyano-3-(methylsulfonyl)-3-(1,2,4,5-tetrahydro-3H-3-benzazepin-3-yl-2-propenoicacid;(2,2-bis[p-(dimethylamino)phenyl]vinyl](methylsulfonyl)-fumaronitrile;[2,2-bis[p-dimethylamino)phenyl]vinyl](hydroxymethyl)sulfonyl)-fumaronitrile,p-toluenesulfonate;[2,2-bis[p-dimethylamino)phenyl]vinyl](ethylsulfonyl)-fumaronitrile;(benzylsulfonyl) [2,2-bis[p-(dimethylamino)phenyl]vinyl]-fumaronitrile;(allylsulfonyl) [2,2-bis[p-dimethylamino)phenyl]vinyl]-fumaronitrile;benzyl(benzylsulfonyl)-fumaronitrile;1,7-bis(allylsulfonyl)-4-hydroxy-1,3,5-Heptatriene-1,2,6,7-tetracarbonitrile;2-cyano-3-(methylsulfonyl)-3-(1,2,4,5-tetrahydro-3H-3-benzazepin-3-yl)-2-propenoicacid ethyl ester;alpha-[[[(1E)-1-cyano-2-(3,4-dihydroxyphenyl)ethenyl]sulfonyl]methylene]-3,4-dihydroxy-beta-oxo-(alphaE)-benzenepropanenitrile; 3-(methylsulfonyl)-(2E)-2-propenenitrile;4,4,4-trifluoro-3-(hexylsulfonyl)-(2Z)-2-butenenitrile;alpha-[[[1-cyano-3-(3,4-dihydroxyphenyl)-3-oxo-1-propenyl]sulfonyl]methylene]-3,4-dihydroxy-(E,E)-benzeneacetonitrile;2,2′-[1,3-propanediylbis[(cyclohexylimino)4,1-phenylene]]bis[3-(ethylsulfonyl)-2-butenedinitrile;alpha-(methylsulfonyl)methylene)-(Z)-benzeneacetonitrile;alpha-[[(1,1-dimethylethyl)sulfonyl]methylene]-(Z)-benzeneacetonitrile;alpha-[methylsulfonyl)methylene]-(E)-benzeneacetonitrile;alpha-[[(phenylmethyl)sulfonyl]methylene]-benzeneacetonitrile;alpha-[(butylsulfonyl)methylene]-(E)-benzeneacetonitrile;alpha-[(butylsulfonyl)methylene)-(Z)-benzeneacetonitrile;alpha-[[(1-methylethyl)sulfonyl]methylene]-(E)-benzeneacetonitrile;alpha-[[(1-methylethy])sulfonyl]methylene]-(Z)-benzeneacetonitrile;alpha-[[(1,1-dimethylethyl)sulfonyl]methylene]-(E)-benzeneacetonitrile;alpha-[[[4-chlorophenyl)methyl]sulfonyl]methylene]-(E)-benzeneacetonitrile;alpha-[[[4-chlorophenyl)methyl]sulfonyl]methylene]-(Z)-benzeneacetonitrile;alpha-[[3-chloropropyl)sulfonyl]methylene]-(E)-benzeneacetonitrile;alpha-[[3-chloropropyl)sulfonyl]methylene]-(Z)-benzeneacetonitrile;3-(methylsulfonyl)-(2E)-2-propenenitrile;3-(methylsulfonyl)-(Z)-2-propenenitrile;alpha-[(methylsulfonyl)methylene]-2-nitro-benzeneacetonitrile;4-(dimethylamino)-alpha-[[(trifluoromethyl)sulfonyl]methylene]-benzeneacetonitrile;2-chloro-3-(methylsulfonyl)-2-propenenitrile;2,3-dichloro-3-(methylsulfonyl)-2-propenenitrile;2,3-dichloro-3-[(1-methylethyl)sulfonyl]-2-propenenitrile;2,3-dichloro-3-[(1-methylpropyl)sulfonyl]-2-propenenitrile;2,3-dichloro-3-[(3-methylbutyl)sulfonyl]-2-propenenitrile;2,3-dichloro-3-(octylsulfonyl)-2-propenenitrile;2,3-dichloro-3-(nonylsulfonyl)-2-propenenitrile;3-[(2-phenylethenyl)sulfonyl)-2-propenenitrile;3-[(2-phenylethenyl)sulfony1]-2-propenenitrile;alpha-[methylsulfonyl)phenylmethylene]-(Z)-benzeneacetonitrile;3-(benzylsulfonyl)-acrylonitrile; 3-(methylsulfonyl)-2-propenenitrile;3-(ethylsulfonyl)-acrylonitrile;3,3′-(tetramethylenedisulfonyl)di-acrylonitrile;3-[(2-cyanovinyl)sulfonyl)-propionitrile;3-((4-(2,2-dichloro-1,1-difluoroethoxy)phenyl)sulfonyl)-2-propenenitrile;3-((4-(2,2-dichloro-1,1-difluoroethoxy)-2-methyl-5-nitropheny)sulfonyl)-2-propenenitrile;or 3-((3-trifluoromethyl)phenyl)sulfonyl)-2-propenenitrile.

In another embodiment, the inhibitor is(2E)-3-(tolysulfonyl)-2-propenenitrile, which is also referred to asBAY-7082.

In another embodiment, the inhibitor is (2E)-3-[4-(tertiary butylphenyl)sulfonyl]-2-propenenitrile, which is also referred to asBAY-11-7085.

In an alternative embodiment, the inhibitors useful in any of themethods comprise at least one amino acid residue. An “amino acidresidue” is produced when an amino acid is reacted with one or morecompounds capable of reacting with the amino acid. For example, when anamino acid is reacted with two other amino acids to produce atripeptide, the resultant tripeptide contains three amino acid residues.Alternatively, the amino acid can react with other non-amino acids toproduce a compound having an amino acid residue.

In one embodiment, the inhibitor has at least one leucine residue. Inanother embodiment, the inhibitor comprises three leucine residues. In afurther embodiment, the compound isN-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide,which has the structure

This compound is also referred to as Sigma MG-132.

In another embodiment, the inhibitor is pyrrolidine dithiocarbamate,which has the structure II

wherein R₆ and R₇ are, independently, hydrogen, alkyl, alkenyl, alkynyl,aralkyl, or substituted or unsubstituted aromatic, or R₆ and R₇ togetherform a ring with the nitrogen atom, and

-   -   X and Y are, independently, oxygen or sulfur,    -   or the pharmaceutically-accpetable salt, ester, or amide        thereof.

In one embodiment, X and Y are both oxygen. In another embodiment, X andY are both sulfur. In a further embodiment, R₆ and R₇ can form a ringsystem. For example, R₆ and R₇ can be collectively a methylene groupsuch as (CH₂)₃. The resultant ring structure would be a four-memberedring with one nitrogen atom. When R₆ and R₇ form a ring, the ring can befrom a three- to 10-membered ring. In one embodiment, X and Y aresulfur, and R₆ and R₇ is (CH₂)₄. This compound is also referred toherein as PDTC.

Formula II also enompasses pharmaceutically acceptable esters, amides,and salts of such compounds. Pharmaceutically acceptable salts areprepared by treating the free acid with an appropriate amount of apharmaceutically acceptable base. Representative pharmaceuticallyacceptable bases are ammonium hydroxide, sodium hydroxide, potassiumhydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide,ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide,ferric hydroxide, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, lysine, arginine, histidine, and the like. In oneaspect, the reaction is conducted in water, alone or in combination withan inert, water-miscible organic solvent, at a temperature of from about0° C. to about 100° C. such as at room temperature. The molar ratio ofcompounds of structural formula II to base used are chosen to providethe ratio desired for any particular salts. For preparing, for example,the ammonium salts of the free acid starting material, the startingmaterial can be treated with approximately one equivalent ofpharmaceutically acceptable base to yield a neutral salt.

Ester derivatives are typically prepared as precursors to the acid formof the compounds—as illustrated in the examples below—and accordinglycan serve as prodrugs. Generally, these derivatives will be lower alkylesters such as methyl, ethyl, and the like. Amide derivatives —(CO)NH₂,—(CO)NHR and —(CO)NR₂, where R is an alkyl group defined above, can beprepared by reaction of the carboxylic acid-containing compound withammonia or a substituted amine.

a) Direct Inhibitors

Disclosed are inhibitors which are direct inhibitors of NF-κB. A directinhibitor of NF-κB is an inhibitor that interacts with NF-κB. A directinhibitor touches in some way the NF-κB molecule such that the NF-κBdependent activities, such as readhesion and metastasis are inhibited.

b) Indirect Inhibitors

Disclosed are inhibitors which are indirect inhibitors of NF-κB. Anindirect inhibitor of NF-κB is an inhibitor that does not interact withNF-κB. An indirect inhibitor touches in some way a molecule that isinvolved in a signal transduction pathway that NF-κB is involved in suchthat the NF-κB dependent activities, such as readhesion and metastasisare inhibited.

One example of an indirect inhibitor of NF-κB would be an indirectinhibitor which inhibits the expression of NF-κB and thereby preventNF-κB from functioning. Another example of an indirect inhibitor ofNF-κB would be an indirect inhibitor that inhibits translation of a geneencoding NF-κB.

An example of a molecule that is involved in a signal transductionpathway that NF-κB is involved in is IκB. Molecules that inhibit IκBfrom phosphorylating can be indirect inhibitors.

2. Homology/Identity

It is understood that one way to define any known variants andderivatives or those that might arise, of the disclosed genes andproteins herein is through defining the variants and derivatives interms of homology to specific known sequences. For example, thesequences of a particular human NF-κB and IκB are known and readilyobtainable at for example, a sequence database such as Genbank. Thenucleic acids that encode these proteins are also readily available.These sequences and any known alleles or species variants are considereddisclosed herein. Specifically disclosed are variants of these and othergenes and proteins herein disclosed which have at least, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 percent homology to the stated sequence.Those of skill in the art readily understand how to determine thehomology of two proteins or nucleic acids, such as genes. For example,the homology can be calculated after aligning the two sequences so thatthe homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

3. Hybridization/Selective Hybridization

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a gene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference for material at least related to hybridizationof nucleic acids). A preferable stringent hybridization condition for aDNA:DNA hybridization can be at about 68° C. (in aqueous solution) in6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingprimer is in for example, 10 or 100 or 1000 fold excess. This type ofassay can be performed at under conditions where both the limiting andnon-limiting primer are for example, 10 fold or 100 fold or 1000 foldbelow their k_(d), or where only one of the nucleic acid molecules is 10fold or 100 fold or 1000 fold or where one or both nucleic acidmolecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of primer that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer isenzymatically manipulated under conditions which promote the enzymaticmanipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer molecules are extended. Preferred conditions alsoinclude those suggested by the manufacturer or indicated in the art asbeing appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein.

4. Nucleic Acids

There are a variety of molecules disclosed herein that are nucleic acidbased, including for example the nucleic acids that encode, for exampleNF-κB or IκB, as well as various functional nucleic acids. The disclosednucleic acids are made up of for example, nucleotides, nucleotideanalogs, or nucleotide substitutes. Non-limiting examples of these andother molecules are discussed herein. It is understood that for example,when a vector is expressed in a cell, that the expressed mRNA willtypically be made up of A, C, G, and U. Likewise, it is understood thatif, for example, an antisense molecule is introduced into a cell or cellenvironment through for example exogenous delivery, it is advantagousthat the antisense molecule be made up of nucleotide analogs that reducethe degradation of the antisense molecule in the cellular environment.

a) Nucleotides and Related Molecules

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Annon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989,86, 6553-6556),

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or O) at the C6 position of purinenucleotides.

b) Sequences

There are a variety of sequences related to for example, the NF-κB orIκB genes, found in sequence data bases, such as Genbank. Thesesequences and others are herein incorporated by reference in theirentireties as well as for individual subsequences contained therein.

Those of skill in the art understand how to resolve sequencediscrepancies and differences and to adjust the compositions and methodsrelating to a particular sequence to other related sequences (i.e.sequences of NF-κB or IκB). Primers and/or probes can be designed forany NF-κB or IκB sequence given the information disclosed herein andknown in the art.

c) Primers and Probes

Disclosed are compositions including primers and probes, which arecapable of interacting with, for example, the NF-κB or IκB nucleicacids, such as mRNA, as disclosed herein. In certain embodiments theprimers are used to support DNA amplification reactions. Typically theprimers will be capable of being extended in a sequence specific manner.Extension of a primer in a sequence specific manner includes any methodswherein the sequence and/or composition of the nucleic acid molecule towhich the primer is hybridized or otherwise associated directs orinfluences the composition or sequence of the product produced by theextension of the primer. Extension of the primer in a sequence specificmanner therefore includes, but is not limited to, PCR, DNA sequencing,DNA extension, DNA polymerization, RNA transcription, or reversetranscription. Techniques and conditions that amplify the primer in asequence specific manner are preferred. In certain embodiments theprimers are used for the DNA amplification reactions, such as PCR ordirect sequencing. It is understood that in certain embodiments theprimers can also be extended using non-enzymatic techniques, where forexample, the nucleotides or oligonucleotides used to extend the primerare modified such that they will chemically react to extend the primerin a sequence specific manner. Typically the disclosed primers hybridizewith, for example, the NF-κB nucleic acid, such as mRNA, or the IκBnucleic acid, such as mRNA, or region of the NF-κB or IκB nucleic acidsor they hybridize with the complement of the NF-κB or IκB nucleic acidsor complement of a region of the NF-κB or IκB nucleic acids.

d) Functional Nucleic Acids

Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can be divided into thefollowing categories, which are not meant to be limiting. For example,functional nucleic acids include antisense molecules, aptamers,ribozymes, triplex forming molecules, and external guide sequences. Thefunctional nucleic acid molecules can act as affectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules can possess a de novoactivity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA of NF-κB or IκB or the genomicDNA of NF-κB or IκB or they can interact with the polypeptide of NF-κBor IκB. Often functional nucleic acids are designed to interact withother nucleic acids based on sequence homology between the targetmolecule and the functional nucleic acid molecule. In other situations,the specific recognition between the functional nucleic acid moleculeand the target molecule is not based on sequence homology between thefunctional nucleic acid molecule and the target molecule, but rather isbased on the formation of tertiary structure that allows specificrecognition to take place. Both of these recognition motifs can alsooccur in the same functional nucleic acid molecule.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule exist. Exemplary methods would be in vitro selectionexperiments and DNA modification studies using DMS and DEPC. It ispreferred that antisense molecules bind the target molecule with adissociation constant (k_(d)) less than 10⁻⁶. It is more preferred thatantisense molecules bind with a k_(d) less than 10⁻⁸. It is also morepreferred that the antisense molecules bind the target molecule with ak_(d) less than 10⁻¹⁰. It is also preferred that the antisense moleculesbind the target molecule with a k_(d) less than 10⁻¹². A representativesample of methods and techniques which aid in the design and use ofantisense molecules can be found in the following non-limiting list ofU.S. Pat. Nos. 5,135,917, 5,294,533, 5,627,158,5,641,754, 5,691,317,5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590,5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522,6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004,6,046,319, and 6,057,437.

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S.Pat. No. 5,580,737), as well as large molecules, such as reversetranscriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No.5,543,293). Aptamers can bind very tightly with k_(d)s from the targetmolecule of less than 10⁻¹² M. It is preferred that the aptamers bindthe target molecule with a k_(d) less than 10⁻⁸. It is more preferredthat the aptamers bind the target molecule with a k_(d) less than 10⁻⁸.It is also more preferred that the aptamers bind the target moleculewith a k_(d) less than 10⁻¹⁸. It is also preferred that the aptamersbind the target molecule with a k_(d) less than 10⁻¹². Aptamers can bindthe target molecule with a very high degree of specificity. For example,aptamers have been isolated that have greater than a 10000 folddifference in binding affinities between the target molecule and anothermolecule that differ at only a single position on the molecule (U.S.Pat. No. 5,543,293). It is preferred that the aptamer have a k_(d) withthe target molecule at least 10 fold lower than the k_(d) with abackground binding molecule. It is more preferred that the aptamer havea k_(d) with the target molecule at least 100 fold lower than the k_(d)with a background binding molecule. It is more preferred that theaptamer have a k_(d) with the target molecule at least 1000 fold lowerthan the k_(d) with a background binding molecule. It is preferred thatthe aptamer have a k_(d) with the target molecule at least 10000 foldlower than the k_(d) with a background binding molecule. It is preferredwhen doing the comparison for a polypeptide for example, that thebackground molecule be a different polypeptide. For example, whendetermining the specificity of NF-κB or IκB aptamers, the backgroundprotein could be serum albumin. Representative examples of how to makeand use aptamers to bind a variety of different target molecules can befound in the following non-limiting list of U.S. Pat. Nos. 5,476,766,5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721,5,846,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691,6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and6,051,698.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly.Ribozymes are thus catalytic nucleic acid. It is preferred that theribozymes catalyze intermolecular reactions. There are a number ofdifferent types of ribozymes that catalyze nuclease or nucleic acidpolymerase type reactions which are based on ribozymes found in naturalsystems, such as hammerhead ribozymes, (for example, but not limited tothe following U.S. Pat. Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133,5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288,5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but notlimited to the following U.S. Pat. Nos. 5,631,115, 5,646,031, 5,683,902,5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), andtetrahymena ribozymes (for example, but not limited to the followingU.S. Pat. Nos. 5,595,873 and 5,652,107). There are also a number ofribozymes that are not found in natural systems, but which have beenengineered to catalyze specific reactions de novo (for example, but notlimited to the following U.S. Pat. Nos. 5,580,967, 5,688,670, 5,807,718,and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, andmore preferably cleave RNA substrates. Ribozymes typically cleavenucleic acid substrates through recognition and binding of the targetsubstrate with subsequent cleavage. This recognition is often basedmostly on canonical or non-canonical base pair interactions. Thisproperty makes ribozymes particularly good candidates for targetspecific cleavage of nucleic acids because recognition of the targetsubstrate is based on the target substrates sequence. Representativeexamples of how to make and use ribozymes to catalyze a variety ofdifferent reactions can be found in the following non-limiting list ofU.S. Pat. Nos. 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855,5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and6,017,756.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed, in which there are three strands of DNA forming acomplex dependant on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a k_(d) less than 10⁻⁶.It is more preferred that the triplex forming molecules bind with ak_(d) less than 10⁻⁸. It is also more preferred that the triplex formingmolecules bind the target molecule with a k_(d) less than 10⁻¹⁰. It isalso preferred that the triplex forming molecules bind the targetmolecule with a k_(d) less than 10⁻¹². Representative examples of how tomake and use triplex forming molecules to bind a variety of differenttarget molecules can be found in the following non-limiting list of U.S.Pat. Nos. 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773,5,834,185, 5,869,246, 5,874,566, and 5,962,426.

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNaseP, which cleaves the target molecule. EGSs can be designed tospecifically target a RNA molecule of choice. RNAse P aids in processingtransfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited tocleave virtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 byYale, and Forster and Altman, Science 238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can beutilized to cleave desired targets within eukarotic cells. (Yuan et al.,Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO95/24489 by Yale; Yuan and Altman, EMBO J 14:159-168 (1995), and Carraraet al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)).Representative examples of how to make and use EGS molecules tofacilitate cleavage of a variety of different target molecules be foundin the following non-limiting list of U.S. Pat. Nos. 5,168,053,5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162

5. Peptides a) Protein Variants

As discussed herein there are numerous variants of the NF-κB protein andIκB protein that are known and herein contemplated. Protein variants andderivatives are well understood to those of skill in the art and in caninvolve amino acid sequence modifications. For example, amino acidsequence modifications typically fall into one or more of three classes:substitutional, insertional or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.Immunogenic fusion protein derivatives, such as those described in theexamples, are made by fusing a polypeptide sufficiently large to conferimmunogenicity to the target sequence by cross-linking in vitro or byrecombinant cell culture transformed with DNA encoding the fusion.Deletions are characterized by the removal of one or more amino acidresidues from the protein sequence. Typically, no more than about from 2to 6 residues are deleted at any one site within the protein molecule.These variants ordinarily are prepared by site specific mutagenesis ofnucleotides in the DNA encoding the protein, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample M13 primer mutagenesis and PCR mutagenesis. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables 1 and 2 and are referred to as conservative substitutions. TABLE1 Amino Acid Abbreviations Amino Acid Abbreviations Alanine AlaAAllosoleucine AIle Arginine ArgR Asparagines AsnN aspartic acid AspDCysteine CysC glutamic acid GluE Glutamine GlnK Glycine GlyG HistidineHisH Isolelucine IleI Leucine LeuL Lysine LysK Phenylalanine PheFProline ProP pyroglutamic acidp Glu Serine SerS Threonine ThrT TyrosineTyrY Tryptophan TrpW Valine ValV

TABLE 2 Amino Acid Substitutions Exemplary Conservative Substitutions,others are Original Residue known in the art. Ala ser Arg lys, gln Asngln; his Asp glu Cys ser Gln asn, lys Glu asp Gly ala His asn; gln Ileleu; val Leu ile; val Lys arg; gln; Met Leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Va lile; leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table2, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[19831]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

6. Antibodies a) Antibodies Generally

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof, aslong as they are chosen for their ability to interact with NF-κB or IκBsuch that NF-κB or IκB are inhibited from causing the cellularproliferative events disclosed herein. The antibodies can be tested fortheir desired activity using the in vitro assays described herein, or byanalogous methods, after which their in vivo therapeutic and/orprophylactic activities are tested according to known clinical testingmethods. Also disclosed are functional equivalents of antibodies.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch, antibodies, as long as they exhibit the desired antagonisticactivity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedurewhich produces mono clonal antibodies. For example, monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNAencoding the disclosed monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. No. 5,804,440 toBurton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin.Biotechnol. 3:348-354, 1992).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse.

b) Human Antibodies

The human antibodies can be prepared using any technique. Examples oftechniques for human monoclonal antibody production include thosedescribed by Cole et al. (Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol., 147 (1):86-95,1991). Human antibodies (and fragments thereof) can also be producedusing phage display libraries (Hoogenboom et al., J. Mol. Biol.,227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991).

The human antibodies can also be obtained from transgenic animals. Forexample, transgenic, mutant mice that are capable of producing a fullrepertoire of human antibodies, in response to immunization, have beendescribed (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA,90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, thehomozygous deletion of the antibody heavy chain joining region (J(H))gene in these chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production, and the successfultransfer of the human germ-line antibody gene array into such germ-linemutant mice results in the production of human antibodies upon antigenchallenge.

c) Humanized Antibodies

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

d) Administration of Antibodies

Administration of the antibodies can be done as disclosed herein:Nucleic acid approaches for antibody delivery also exist. The broadlyneutralizing anti NF-κB or IκB antibodies and antibody fragments canalso be administered to patients or subjects as a nucleic acidpreparation (e.g., DNA or RNA) that encodes the antibody or antibodyfragment, such that the patient's or subject's own cells take up thenucleic acid and produce and secrete the encoded antibody or antibodyfragment. The delivery of the nucleic acid can be by any means, asdisclosed herein, for example.

7. Delivery of the Compositions to Cells a) Nucleic Acid Delivery

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991) Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modified to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

In the methods described herein, which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids can be in the form ofnaked DNA or RNA, or the nucleic acids can be in a vector for deliveringthe nucleic acids to the cells, whereby the encoding DNA or DNA orfragment is under the transcriptional regulation of a promoter, as wouldbe well understood by one of ordinary skill in the art as well asenhancers. The vector can be a commercially available preparation, suchas an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec,Canada).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486,1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells nucleic acid encoding a broadly neutralizing antibody (oractive fragment thereof). The exact method of introducing the alterednucleic acid into mammalian cells is, of course, not limited to the useof retroviral vectors. Other techniques are widely available for thisprocedure including the use of adenoviral vectors (Mitani et al., Hum.Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors(Goodman et al., Blood 84:1492-1500, 1994), lentiviral vectors (Naidiniet al., Science 272:263-267, 1996), pseudotyped retroviral vectors(Agrawal et al., Exper. Hematol. 24:738-747, 1996). Physicaltransduction techniques can also be used, such as liposome delivery andreceptor-mediated and other endocytosis mechanisms (see, for example,Schwartzenberger et al., Blood 87:472-478, 1996). The disclosedcompositions and methods can be used in conjunction with any of these orother commonly used gene transfer methods.

As one example, if the antibody-encoding nucleic acid or some othernucleic acid encoding an inhibitor of the NF-κB or IκB proteins orencoding a particular variant of the NF-κB or IκB genes to be used inthe disclosed methods, is delivered to the cells of a subject in anadenovirus vector, the dosage for administration of adenovirus to humanscan range from about 10⁷ to 10⁹ plaque forming units (pfu) per injectionbut can be as high as 10¹² pfu per injection (Crystal, Hum. Gene Ther.8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997).A subject can receive a single injection, or, if additional injectionsare necessary, they can be repeated at six month intervals (or otherappropriate time intervals, as determined by the skilled practitioner)for an indefinite period and/or until the efficacy of the treatment hasbeen established.

Parenteral administration of the nucleic acid or vector, if used, isgenerally characterized by injection. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution of suspension in liquid prior to injection,or as emulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein. For additionaldiscussion of suitable formulations and various routes of administrationof therapeutic compounds, see, e.g., Remington: The Science and Practiceof Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,Easton, Pa. 1995.

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral integration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can become integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

b) Non-Nucleic Acid Based Systems

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosedcompositions or vectors for example, lipids such as liposomes, such ascationic liposomes (e.g., DOTMA, s DOPE, DC-cholesterol) or anionicliposomes. Liposomes can further comprise proteins to facilitatetargeting a particular cell, if desired. Administration of a compositioncomprising a compound and a cationic liposome can be administered to theblood afferent to a target organ or inhaled into the respiratory tractto target cells of the respiratory tract. Regarding liposomes, see,e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989);Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat.No. 4,897,355. Furthermore, the compound can be administered as acomponent of a microcapsule that can be targeted to specific cell types,such as macrophages, or where the diffusion of the compound or deliveryof the compound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the nucleicacid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Inc. (San Diego,Calif.) as well as by means of a SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). These techniques can be used for avariety of other specific cell types. Vehicles such as “stealth” andother antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

c) In Vivo/Ex Vivo

As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to the subjectscells in vivo and/or ex vivo by a variety of mechanisms well known inthe art (e.g., uptake of naked DNA, liposome fusion, intramuscularinjection of DNA via a gene gun, endocytosis and the like).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

8. Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

a) Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, -fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promoter and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTF.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

b) Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

9. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,although topical intranasal administration or administration by inhalantis typically preferred. As used herein, “topical intranasaladministration” means delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization of the nucleic acid or vector. The latter may beeffective when a large number of animals is to be treatedsimultaneously. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

b) Therapeutic Uses

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptomsdisorder are effected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

c) Combinatorial Chemistry

The disclosed compositions can be used as targets for any combinatorialtechnique to identify molecules or macromolecular molecules thatinteract with the disclosed compositions in a desired way. Alsodisclosed are the compositions that are identified through combinatorialtechniques or screening techniques in which the compositions interactwith NF-κB or IκB such that the compositions decrease the cellularproliferative activity of NF-κB and IκB or portions thereof, where thecompositions were identified using NF-κB or IκB as targets in ascreening or selection protocol.

It is understood that when using the disclosed compositions incombinatorial techniques or screening methods, molecules, such asmacromolecular molecules, will be identified that have particulardesired properties such as inhibition or stimulation or the targetmolecule's function. The molecules identified and isolated when usingthe disclosed compositions, such as, NF-κB or IκB are used as targets,or when the disclosed compositions like BAY-11-7082 or BAY-11-7085 areused in competitive inhibition assays are also disclosed. Thus, theproducts produced using the combinatorial or screening approaches thatinvolve the disclosed compositions are also considered herein disclosed.

Combinatorial chemistry includes but is not limited to all methods forisolating small molecules or macromolecules that are capable of bindingeither a small molecule or another macromolecule, typically in aniterative process. Proteins, oligonuclecotides, and sugars are examplesof macromolecules. For example, oligonucleotide molecules with a givenfunction, catalytic or ligand-binding, can be isolated from a complexmixture of random oligonucleotides in what has been referred to as “invitro genetics” (Szostak, TIBS 19:89, 1992). One synthesizes a largepool of molecules bearing random and defined sequences and subjects thatcomplex mixture, for example, approximately 10¹⁵ individual sequences in100 μg of a 100 nucleotide RNA, to some selection and enrichmentprocess. Through repeated cycles of affinity chromatography and PCRamplification of the molecules bound to the ligand on the column,Ellington and Szostak (1990) estimated that 1 in 10¹⁰ RNA moleculesfolded in such a way as to bind a small molecule dyes. DNA moleculeswith such ligand-binding behavior have been isolated as well (Ellingtonand Szostak, 1992; Bock et al, 1992). Techniques aimed at similar goalsexist for small organic molecules, proteins, antibodies and othermacromolecules known to those of skill in the art. Screening sets ofmolecules for a desired activity whether based on small organiclibraries, oligonucleotides, or antibodies is broadly referred to ascombinatorial chemistry. Combinatorial techniques are particularlysuited for defining binding interactions between molecules and forisolating molecules that have a specific binding activity, often calledaptamers when the macromolecules are nucleic acids.

There are a number of methods for isolating proteins which either havede novo activity or a modified activity. For example, phage displaylibraries have been used to isolate numerous peptides that interact witha specific target. (See for example, U.S. Pat. Nos. 6,031,071;5,824,520; 5,596,079; and 5,565,332 which are herein incorporated byreference at least for their material-related to phage display andmethods relate to combinatorial chemistry).

A preferred method for isolating proteins that have a given function isdescribed by Roberts and Szostak (Roberts R. W. and Szostak J. W. Proc.Natl. Acad. Sci. USA, 94(23)12997-302 (1997). This combinatorialchemistry method couples the functional power of proteins and thegenetic power of nucleic acids. An RNA molecule is generated in which apuromycin molecule is covalently attached to the 3′-end of the RNAmolecule. An in vitro translation of this modified RNA molecule causesthe correct protein, encoded by the RNA to be translated. In addition,because of the attachment of the puromycin, a peptdyl acceptor whichcannot be extended, the growing peptide chain is attached to thepuromycin which is attached to the RNA. Thus, the protein molecule isattached to the genetic material that encodes it. Normal in vitroselection procedures can now be done to isolate functional peptides.Once the selection procedure for peptide function is completetraditional nucleic acid manipulation procedures are performed toamplify the nucleic acid that codes for the selected functionalpeptides. After amplification of the genetic material, new RNA istranscribed with puromycin at the 3′-end, new peptide is translated andanother functional round of selection is performed. Thus, proteinselection can be performed in an iterative manner just like nucleic acidselection techniques. The peptide which is translated is controlled bythe sequence of the RNA attached to the puromycin. This sequence can beanything from a random sequence engineered for optimum translation (i.e.no stop codons etc.) or it can be a degenerate sequence of a known RNAmolecule to look for improved or altered function of a known peptide.The conditions for nucleic acid amplification and in vitro translationare well known to those of ordinary skill in the art and are preferablyperformed as in Roberts and Szostak (Roberts R. W. and Szostak J. W.Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997)).

Another preferred method for combinatorial methods designed to isolatepeptides is described in Cohen et al. (Cohen B. A.,et al., Proc. Natl.Acad. Sci. USA 95(24):14272-7 (1998)). This method utilizes and modifiestwo-hybrid technology. Yeast two-hybrid systems are useful for thedetection and analysis of protein:protein interactions. The two-hybridsystem, initially described in the yeast Saccharomyces cerevisiae, is apowerful molecular genetic technique for identifying new regulatorymolecules, specific to the protein of interest (Fields and Song, Nature340:245-6 (1989)). Cohen et al., modified this technology so that novelinteractions between synthetic or engineered peptide sequences could beidentified which bind a molecule of choice. The benefit of this type oftechnology is that the selection is done in an intracellularenvironment. The method utilizes a library of peptide molecules thatattached to an acidic activation domain. A peptide of choice, forexample a portion of NF-κB or IκB is attached to a DNA binding domain ofa transcriptional activation protein, such as Gal 4. By performing thetwo-hybrid technique on this type of system, molecules that bind desiredfragments of NF-κB or IκB can be identified.

Using methodology well known to those of skill in the art, incombination with various combinatorial libraries, one can isolate andcharacterize those small molecules or macromolecules, which bind to orinteract with the desired target. The relative binding affinity of thesecompounds can be compared and optimum compounds identified usingcompetitive binding studies, which are well known to those of skill inthe art.

Techniques for making combinatorial libraries and screeningcombinatorial libraries to isolate molecules which bind a desired targetare well known to those of skill in the art. Representative techniquesand methods can be found in but are not limited to U.S. Pat. Nos.5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083, 5,545,568,5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825, 5,619,680,5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899,5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099, 5,723,598,5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130, 5,831,014,5,834,195, 5,834,318, 5,834,588, 5,840,500, 5,847,150, 5,856,107,5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214, 5,880,972,5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955, 5,925,527,5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702, 5,958,792,5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704, 5,985,356,5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768, 6,025,371,6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and 6,061,636.

Combinatorial libraries can be made from a wide array of molecules usinga number of different synthetic techniques. For example, librariescontaining fused 2,4-pyrimidinediones (U.S. Pat. No. 6,025,371)dihydrobenzopyrans (U.S. Pat. Nos. 6,017,768 and 5,821,130), amidealcohols (U.S. Pat. No. 5,976,894), hydroxy-amino acid amides (U.S. Pat.No. 5,972,719) carbohydrates (U.S. Pat. No. 5,965,719),1,4-benzodiazepin-2,5-diones (U.S. Pat. No. 5,962,337), cyclics (U.S.Pat. No. 5,958,792), biaryl amino acid amides (U.S. Pat. No. 5,948,696),thiophenes (U.S. Pat. No. 5,942,387), tricyclic Tetrahydroquinolines(U.S. Pat. No. 5,925,527), benzofurans (U.S. Pat. No. 5,919,955),isoquinolines (U.S. Pat. No. 5,916,899), hydantoin and thiohydantoin(U.S. Pat. No. 5,859,190), indoles (U.S. Pat. No. 5,856,496),imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (U.S. Pat.No. 5,856,107) substituted 2-methylene-2,3-dihydrothiazoles (U.S. Pat.No. 5,847,150), quinolines (U.S. Pat. No. 5,840,500), PNA (U.S. Pat. No.5,831,014), containing tags (U.S. Pat. No. 5,721,099), polyketides (U.S.Pat. No. 5,712,146), morpholino-subunits (U.S. Pat. Nos. 5,698,685 and5,506,337), sulfamides (U.S. Pat. No. 5,618,825), and benzodiazepines(U.S. Pat. No. 5,288,514).

As used herein combinatorial methods and libraries included traditionalscreening methods and libraries as well as methods and libraries used initerative processes.

d) Computer Assisted Drug Design

The disclosed compositions can be used as targets for any molecularmodeling technique to identify either the structure of the disclosedcompositions or to identify potential or actual molecules, such as smallmolecules, which interact in a desired way with the disclosedcompositions.

It is understood that when using the disclosed compositions in modelingtechniques, molecules, such as macromolecular molecules, will beidentified that have particular desired properties such as inhibition orstimulation or the target molecule's function. The molecules identifiedand isolated when using the disclosed compositions are also disclosed.Thus, the products produced using the molecular modeling approaches thatinvolve the disclosed compositions are also considered herein disclosed.

10. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include molecules, including forexample, BAY 11-7082 or BAY 11-7085 for use in in vitro cell assays asstandards for anti-proliferative activity.

11. Compositions with Similar Functions

It is understood that the compositions, such as BAY 11-7082 and BAY11-7085, disclosed herein have certain functions, such as antimetastaticactivities or anti-proliferative activities. Disclosed herein arecertain structural requirements for performing the disclosed functions,and it is understood that there are a variety of structures which canperform the same function which are related to the disclosed structures,and that these structures will ultimately achieve the same result, forexample, inhibition of anti-proliferative activities.

E. METHODS OF MAKING THE COMPOSITIONS

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

1. Nucleic Acid Synthesis

For example, the nucleic acids, such as, the oligonucleotides to be usedas primers can be made using standard chemical synthesis methods or canbe produced using enzymatic methods or any other known method. Suchmethods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) topurely synthetic methods, for example, by the cyanoethyl phosphoramiditemethod using a Milligen or Beckman System IPlus DNA synthesizer (forexample, Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). Protein nucleic acid molecules can be madeusing known methods such as those described by Nielsen et al.,Bioconjug. Chem. 5:3-7 (1994).

2. Peptide Synthesis

One method of producing the disclosed proteins or polypeptides is tolink two or more peptides or polypeptides together by protein chemistrytechniques. For example, peptides or polypeptides can be chemicallysynthesized using currently available laboratory equipment using eitherFmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to the disclosed proteins, for example, can be synthesizedby standard chemical reactions. For example, a peptide or polypeptidecan be synthesized and not cleaved from its synthesis resin whereas theother fragment of a peptide or protein can be synthesized andsubsequently cleaved from the resin, thereby exposing a terminal groupwhich is functionally blocked on the other fragment. By peptidecondensation reactions, these two fragments can be covalently joined viaa peptide bond at their carboxyl and amino termini, respectively, toform an antibody, or fragment thereof. (Grant Ga. (1992) SyntheticPeptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky Mand Trost B., Ed. (1993) Principles of Peptide Synthesis.Springer-Verlag Inc., NY (which is herein incorporated by reference atleast for material related to peptide synthesis). Alternatively, thepeptide or polypeptide is independently synthesized in vivo as describedherein. Once isolated, these independent peptides or polypeptides may belinked to form a peptide or fragment thereof via similar peptidecondensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide-thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J.Biol.Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry,30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

3. Processes for Making the Compositions

Disclosed are processes for making the compositions as well as makingthe intermediates leading to the compositions. There are a variety ofmethods that can be used for making these compositions, such assynthetic chemical methods and standard molecular biology methods. It isunderstood that the methods of making these and the other disclosedcompositions are specifically disclosed.

Disclosed are cells produced by the process of transforming the cellwith any of the disclosed nucleic acids. Disclosed are cells produced bythe process of transforming the cell with any of the non-naturallyoccurring disclosed nucleic acids.

Disclosed are any of the disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thenon-naturally occurring disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thedisclosed peptides produced by the process of expressing any of thenon-naturally disclosed nucleic acids.

Disclosed are animals produced by the process of transfecting a cellwithin the animal with any of the nucleic acid molecules disclosedherein. Disclosed are animals produced by the process of transfecting acell within the animal any of the nucleic acid molecules disclosedherein, wherein the animal is a mammal. Also disclosed are animalsproduced by the process of transfecting a cell within the animal any ofthe nucleic acid molecules disclosed herein, wherein the mammal ismouse, rat, rabbit, cow, sheep, pig, or primate.

Also disclose are animals produced by the process of adding to theanimal any of the cells disclosed herein.

F. EXAMPLES

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric.

1. Example 1 Materials & Methods a) Materials

The NF-κB inhibitors, BAY 11-7082 (Biomol), BAY 11-7085, (Biomol) andN-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) (Sigma) weresolubilized in DMSO. The antagonistic and chimeric TNFα monoclonalantibody (cA2 ) was purchased from the clinical pharmacy at theUniversity of Utah and reconstituted in sterile water. Polyclonalantibodies to FLIP_(S/L), c-IAP-1, c-IAP-2, TRAF-1, and TRAF-2 wereobtained from Santa Cruz Biotechnology. Antibodies to cleavedpoly(ADP-ribose) polymerase (PARP) were obtained from New EnglandBiolabs. The monoclonal antibody recognizing the p65 NF-κB subunit wasobtained from Santa Cruz. Alkaline phosphatase-conjugatedgoat-anti-rabbit antibody was obtained from Jackson Laboratories.

b) Cell Culture, Proliferation, and Toxicity

DLD-1, HCT-116, and HT-29 colon cancer cell lines were obtained from theATCC collection and were cultured in Dulbecco's Modification of Eagle'sMedium supplemented with 10% fetal bovine serum, glutamine, penicillin,and streptomycin. 293 cells stably transfected with COX-2 (293-COX-2)under the control of a ponasterone sensitive promoter were cultured inDulbecco's Modification of Eagle's Medium supplemented with 10% fetalbovine serum, 400 μg/ml of zeocin, 400 μg/ml of G418, glutamine,penicillin, and streptomycin. For induction of COX-2 protein expression,293-COX-2 cells were cultured for 48 hours in media supplemented with 1μg/ml of ponasterone. All cells were cultured at 37° C. in a humidifiedincubator with a 5% CO₂ atmosphere.

For the cell proliferation assay, cells were dispersed and plated at40,000 cells/well in 96-well dishes. At various days in culture, thecells were gently washed twice with 100 μl/well of ice-cold blockingbuffer (1% radioimmunoassay grade BSA in PBS) and twice with 100 μl/wellof ice-cold PBS. The cells were fixed for 10 min in 100% ice-coldmethanol (100 μl/well), then allowed to air-dry. The cells were stainedwith 100μl/well of 0.1% crystal violet in H₂O for 10 minutes, thenwashed gently four times with dd H₂0 and four times with PBS. The plateswere then air-dried completely. The stained cells were then solubilizedin 1% sodium deoxycholate, and the plates read at 590 nm in aspectrophotometer. The absorption at 590 nm is proportional to thenumber of attached cells.

For the cell toxicity assay, the CytoTox 96® kit from Promega was usedaccording to the manufacturer's instructions. Briefly, DID-1, HCT-116,and HT-29 cells were incubated with 1, 2.5, 5, 7.5 and 10 μM BAY 11-7082or BAY 11-7085 in 96-well plates for 24 hours then lysed by adding 15 μlof lysis 10×Solution (9% (v/v) Triton ® X-100 in water) per 100 μl ofculture medium, followed by incubation at 37° C. for 45-60 minutes.Sample supernatants, (50 μl) were transferred to a fresh 96 wellenzymatic assay plate and incubated with reconstituted Substrate Mix (50μl per sample) for 30 minutes at room temperature in the dark. Theenzymatic assay was then stopped by adding 50 μl/well of Stop Solution.The plate was read at 490 nm and the absorbance values plotted as aratio to the controls (DMSO alone).

c) Electrophoretic Mobility Shift Assay

The adherent cell monolayers were washed once with cold PBS after which200 μl of cold Buffer A (20 mM HEPES pH7.8, 1 mM EDTA, 1 mM EGTA, 10%glycerol, 0.2% NP-40, 0.2 mM Na₃VO₄, 1 mM DTT, 0.5 mM PMSF,and, 1 μg/mleach of aprotinin and leupeptin) was added to the cells. The cells werescraped into microfuge tubes, kept on ice, and sonicated. The lysateswere centrifuged at 500 g for 5 min at 4° C. and the cytoplasmicfraction (supernatant) was removed. 25-50 μl of Buffer B (20 mM HEPES pH7.8, 1 mM EDTA, 1 mM EGTA, 0.42M NaCl, 1.5 mM MgCl₂, 25% glycerol, 0.2mM Na₃VO₄, 1 mM DTT, 0.5 mM PMSF, and, 10 μg/ml each of aprotinin andleupeptin) was added to the nuclear fraction (pellet). The suspensionwas vortexed, kept on ice for 30 minutes, and centrifuged at 13,000 gfor 10 minutes. The supernatants (nuclear fraction) were normalized fortotal protein concentration using the BCA assay (Pierce).

5 pmol of a DNA oligonucleotide containing the NF-κB consensus bindingsite (5′-AGTTGAGGGGACTTTCCCAGGC-3′), 5 μl 5× forward reaction buffer, 10units T4 polynucleotide kinase, 2.5 μl [³²P]ATP (10 μCi/μl, 300Ci/mmol), and water (to 25 μl total volume) were incubated for 10minutes at 37° C. The reaction was stopped by heating the mixture for 10min at 65° C. and the labeled oligonucleotide was separated fromunincorporated [³²P]ATP by centrifuging the mixtures at 12,000-16,000 gfor 30 seconds in G-25 Sepharose columns. The labeled nucleic acid wasrecovered in the collection tube in approximately 25 μl of TE buffer.

For the binding assays, equal amounts of nuclear extracts (approx 2-5 μgof protein) were incubated on ice for 15 minutes with 4 μl 5× gel shiftbinding buffer (50 mM Tris HCl pH 7.5, 250 mM KCl, 5 mM DTT, 1 mg/mlbovine serum albumin, 25% glycerol, H2O (volume adjusted to 5 ml.)), 1.5μg poly (dI-dC), 1 μl ³²P-labeled probe, and enough H₂O to bring thetotal reaction volume to 20 μl. 2 μl 10× loading buffer (20% Glycerol,0.1M Na₂EDTA pH 8, 0.25% bromphenol blue, 0.25% xylene cyanol) was addedto each sample and then loaded onto a native 8% polyacrylamide gel. Thegel was run in 0.5×TBE buffer for about 2 hours, dried, and subjected toautoradiography. The bands on the blots were quantified using the NIHImage program.

Activation of NF-κB was also determined using the Trans-AM™ assay(Active Motif, Carlsbad, Calif.) per the manufacturer's instructions.(Renard et al., 2001) Briefly, cell monolayers on 60 nm dishes werewashed with ice-cold PBS and removed by incubating in trypsin-PBS orscraping. The cells were centrifuged for 10 minutes at 1,000 rpm at 4°C. and resuspended for 10 minutes in 100 μl of 4° C. lysis buffer (20 mMHEPES (pH 7.5), 350 mM NaCl, 20% glycerol, 1% Igepal-CA630, 1 mM MgCl₂,0.5 mM EDTA, 0.1 mM EGTA, 1 μl of 1M dithiothreitol and 10 μl ofprotease inhibitor cocktail (proprietary) per ml of lysis buffer). Thelysates were centrifuged for 20 minutes at 14,000 g at 4° C. Lysatescontaining 5 μg of total protein were added to 20 μl of lysis buffer perwell in 96-well dishes containing immobilized oligonucleotidescorresponding to the NF-κB consensus DNA binding site(5′-GGGACTTTCC-3′). The plate was covered with an adhesive film andincubated for 1 hour at room temperature on a rocker. The wells werethen washed three times with 200 μl per well of washing buffer (100 mMphosphate buffer (pH 7.5), 500 mM NaCl, 1% Tween). 100 μl of p65 subunitmonoclonal antibody (the p65 antibody supplied with the kit onlyrecognizes p65-containing NF-κB heterodimers that are bound to DNAcontaining the NF-κB consensus binding sequence) was diluted 1:1,000 in1× antibody binding buffer (4 mM HEPES (pH7.5), 120 mM KCl, 8% glycerol,1% bovine serum albumin) and added to each well and incubated for 1 hourat room temperature. The wells were washed with 100 μl of washing bufferthree times. 100 μl of horseradish peroxidase-conjugated secondaryantibody diluted 1:1,000 in 1× antibody binding buffer was added to eachwell and incubated for 1 hour at room temperature without agitation. Thewells were washed four times with 200 μl per well of 1× washing buffer.100 μl of developing solution (tetramethylbenzidine in 1% DMSO) wasadded to each well and incubated for 10 minutes at room temperature. 100μl of stop solution (0.5M H₂SO₄) was added to each well after which theabsorbance was determined on a plate spectrophotometer at 450 nm.Specificity of binding was determined using 200-fold excess wildtypeNF-κB oligonucleotides added at the time the cell lysates were added tothe wells.

d) Apoptosis Assay

Single cell suspensions were plated at 100,000 cells per well in 24-wellplates in media containing DMSO, BAY 11-7082, BAY 11-7085, or MG-132 for8-24 hours in an incubator. Alternatively, DMSO, BAY 11-7082, BAY11-7085, or MG-132 were added to cells that were confluent and adherentfor several days. The nonadherent cells were aspirated off with themedia and spun for 3 minutes at 2,000 rpm in 15 ml polypropylene tubes.The adherent cells were washed twice with PBS, dispersed in trypsin, andspun down for 3 minutes at 2,000 rpm in 15 ml polypropylene tubes. Thenonadherent and adherent cells were resuspended in 1× binding buffer (10mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl₂) to a finalconcentration of 10⁶ cells/ml. 100 μl of the cell solution weretransferred to a 5 ml culture tube and incubated with 5 μl of annexinV-FITC-conjugated monoclonal antibody (Pharmingen) and 10 μl ofpropidium iodide (from a 50 μg/ml stock solution made in PBS) in thedark at 25° C. for 15 minutes. 400 μl of binding buffer was added toeach tube and the cells were analyzed by flow cytometry immediately. Thepercent of apoptosis was determined as the percent of annexinV-positive, propidium iodide-negative cells of the total cells counted.

DLD-1 and HT-29 cells that were approximately 60-70% confluent weretransduced with Ad-IκB super-repressor by adding 1-50 μl of purifiedvirus to the cells. After 3 days, the expression of the IκBsuper-repressor was ascertained by immunoblot. In a parallel experiment,the transduced cells were resuspended and allowed to readhere in BAY11-7085 for 8 h. The percentage of surviving (nonapoptotic) cells wasdetermined using crystal violet staining.

e) Immunoblotting

The cells were washed twice in ice cold PBS and then lysed in 4° C.lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 100 mMNaF, 10 mM Na₂PO₄, 1 mM Na₃VO₄, 10% glycerol, 1% Triton X-100, and 1μg/ml each of aprotinin, leupeptin, chymostatin, pepstatin) andclarified at 12,000 rpm at 4° C. for 15 minutes. The lysates werenormalized for total protein concentration. Aliquots of the lysates werethen mixed 1:1 in sample buffer (125 mM Tris-HCl (pH 6.8), 20% glycerol.4% sodium dodecyl sulfate, 2% beta-mercaptoethanol, 10 μg/ml bromophenolblue) and boiled for 3 minutes. The samples were loaded onto SDS-PAGEgels (7.5 or 10% acrylamide). The proteins were transferred tonitrocellulose from the gels, then incubated in blocking buffer (1%bovine serum albumin (Bio-Rad), 100 mM Tris-Cl (pH7.4), 0.9% NaCl, 0.1%Nonidet) overnight at 4° C.

For immunodetection, the blocked blots were probed with primary antibodyfor 2 hours at 4° C. on a rocker. The blots were washed twice for 10minutes each in blocking buffer, then incubated with a 1:2000 dilutionof a rabbit-anti-mouse secondary antibody (for monoclonal primaryantibodies; for polyclonal antibodies, this step was skipped). After twowashes in blocking buffer (150 mM NaCl, 20 mM Tris pH 7.4, 0.5% Tween20, 5% bovine serum albumin, and 0.2 gm/500 ml final concentration ofsodium azide), the blots were finally incubated with a 1:2000 dilutionof alkaline phosphatase-conjugated rabbit-anti-mouse antibodies(Jackson) in blocking buffer. Alkaline phosphatase was detected by acolorimetric reaction using a BCIP (5-Bromo-4-Chloro-3-Indolylphosphate)/NBT (Nitroblue tetrazolium) kit (Zymed).

f) Athymic Mouse Colon Cancer Xenograft Studies

HT-29 and HCT-116 cells were harvested in 0.25% trypsin-PBS-EDTA, washedonce in media and PBS, and resuspended in PBS at 1 million cells per 200μL. One million cells were injected either subcutaneously in the backsor intraperitoneally in 5 week old female nu/nu athymic mice (CharlesRiver Labs). For the mice with subcutaneous tumors, the tumors wereallowed to establish themselves for 10 days after which they wererandomized to BAY 11-7085 or DMSO. For the mice with intraperitonealtumors, the mice received DMSO or BAY 11-7085 (5 mg/kg) 24 hours beforethe cancer cells were injected intrapentoneally. All mice received BAY11-7085 (5 mg/kg) or an equal volume of DMSO (˜200 μL) twice weekly for21-32 days. Subcutaneous tumor sizes were determined by measuring thelength and width with calipers. These studies were approved by the U. ofUtah Institutional Review Board and Institutional Animal Care and UseCommittee and performed in the Animal Resource Center. Mice wereeuthanized when they experienced a greater than 10% loss in body weightor if they appeared ill. Post-mortem examinations included sectioning ofkidney, lung, and liver tissues which were stained with hematoxylin andeosin followed by examination for tissue toxicity/damage by anexperienced mouse pathologist (E.J.E.) who was blinded to therapy.

2. Example 2. Results a) NF-κB Inhibitors Diminish Colon Cancer CellProliferation

Two compounds, BAY 11-7082 and BAY 11-7085, known to inhibit NF-κB andarthritis in rodent models (Pierce et al., 1997) were tested on coloncancer cell lines. BAY 11-7082 and BAY 11-7085 are soluble compoundsthat inhibit IκB phosphorylation and TNFα-induced NF-κB activation.(Pierce et al., 1997) They were also found to activate JNK/SAPK and p38,members of the MAP kinase family of signal transduction proteins.(Pierce et al., 1997) Incubation of the colon cancer cell lines, DLD-1,HCT-116, HT-29, with 10 μM of BAY 11-7085, but not BAY 11-7082 greatlyinhibited cell proliferation (FIGS. 1A & B).

In order to determine whether the differences in the inhibitory effectsof BAY 11-7082 and BAY 11-7085 on colon cancer cell proliferation weredue to differences in the activities of the two compounds, NF-κBelectrophoretic mobility shift assays (EMSA) were performed. Following 3and 6 hour incubations of HT-29 cells with the two compounds at 10 μMconcentrations, a more profound inhibition of NF-κB activity occurredwith BAY 11-7085 than BAY 11-7082 (FIG. 1C). Consistent with previousreports in a variety of cancer cells, HT-29 colon cancer cellsdemonstrated constitutive activation of NF-κB. DLD-1 and HCT-116 cellsdemonstrated constitutive activation of NF-κB by EMSA as well (data notshown).

When HT-29 colon cancer cells were treated with the NF-κB inhibitors,BAY 11-7085 or MG-132 for 24 h, the percentage of apoptotic cells(annexin V-positive and propidium iodide-negative) was relativelygreater for MG-132 than BAY 11-7085, and was proportional to the numberof floating cells. It was confirmed in HT-29 cells that were treatedwith increasing concentrations of BAY 11-7085 for 24 h. After separatingthe HT-29 cells that remained adherent and those that became nonadherentafter BAY 11-7085 treatment, the percentage of apoptotic cells wasdetermined for each group. Whereas the majority of nonadherent cellswere apoptotic, only a minority of the adherent cells was apoptotic. Thepercentage of nonadherent apoptotic cells decreased after treatment with50 and 100 μM BAY 11-7085 compared with lower concentrations of BAY11-7085, because many of the cells were positive for both annexin V andpropridium iodide indicating that they had completed apoptosis and werenow necrotic. Shown another way, HT-29 cells were treated with 20 μMMG-132 for 8 h, after which the adherent and nonadherent cells werecollected separately, and then lysed and immunoblotted for cleaved PARP,a product of caspase cleavage. Whereas there was only a small increasein cleaved PARP in the cells that remained adherent, there was a largeincrease in cleaved PARP in the nonadherent cells. It has been shownthat the loss of colon cancer cell adhesion was associated with theinduction of apoptosis by IFN and TNFα. The inhibition of colon cancercell adhesion appears to be related to the apoptotic effects of theNF-κB inhibitors.

In order to determine whether BAY 11-7082 or BAY 11-7085 causedsignificant direct cell toxicity, an LDH-based cell toxicity assay wasperformed on DLD-1, HCT-116, and HT-29 cell lines. One to ten μM 11-7082or 11-7085 for 24 hours resulted in a maximum of 20% cell toxicity.Thus, direct cell toxicity due to BAY 11-7082 or BAY 11-7085 could notalone explain the decreased colon cancer cell proliferation.

b) NF-κB Inhibitors Inhibit Colon Cancer Cell Tumorigenicity

Anchorage-independent cell growth is a hallmark of cancer cells and is agood correlate to tumorigencity in vivo. Therefore, the effects of BAY11-7082 and BAY 11-7085 were examined on colon cancer cell growth usingthe soft agar colony formation assay. At 10 μM concentrations of BAY11-7082 or BAY 11-7085 for 6 days, anchorage-independent proliferationof the DLD-1 and HT-29, but not HCT-116, colon cancer cell lines wassignificantly inhibited (FIG. 2A).

A recent study showed that inhibition of NF-κB in colon cancer cellxenografts in athymic mice by direct injection of adenovirus containingthe IκB super-repressor mutant into the tumors alone failed to reducethe tumor size. (Wang et al., 1999) However, BAY 11-7082 or BAY 11-7085alone inhibit colon cancer xenograft growth in athymic mice. HC-116, andHT-29 cell lines were used to generate subcutaneous tumors in athymicfemale mice. The mice were subsequently randomized to be treated withvehicle (DMSO) alone or BAY 11-7085 1 mg/kg twice weekly for 28 days.

While HT-29 cells readily formed tumors in the athymic mice, HCT-116cells formed only very small tumors in most cases. There was astatistically significant overall reduction in the tumor volumes in theHT-29 xenografts in the BAY 11-7085 treatment group compared with thecontrol group (FIG. 2B). There was no statistically significant overalldifference in the tumor volumes in the HCT-116 xenografts in thetreatment or control groups (FIG. 2C).

c) NF-κB Inhibitor-Induced Apoptosis is Associated with a Loss andInhibition of Cell Adhesion

Treatment of HT-29 cells with BAY 11-7082 or BAY 11-7085 at doses up to20 μM for 24 hours caused an increase in the level of apoptosis asmeasured by the annexin V-FITC assay (FIG. 3A), which is sensitive forcells in the early stages of apoptosis. However, 20 μM MG-132, anotherNF-κB inhibitor, caused a greater increase in apoptosis (FIG. 3A).Disclosed herein, the level of apoptosis correlated with the degree ofloss of cell adhesion manifested by floating cells. HT-29 cells weretreated with increasing concentrations of BAY 11-7085 for 8 hours, afterwhich the percentage of apoptotic cells was determined separately forthe cells that remained adherent and those that became non-adherent.While the non-adherent cells were predominantly apoptotic, only aminority of the adherent cells was apoptotic (FIG. 3B). Note that thepercentage of apoptosis in the non-adherent cell fraction decreased at50 and 100 μM concentrations of BAY 11-7085. This was due to the factthat after 8 hours of treatment, many of the cells at higher doses ofBAY 11-7085 were annexin V-positive and failed to exclude propidiumiodide meaning that they had completed apoptosis and were now necrotic.Similar results were obtained using another colon cancer cell line,HCT-116, and MG-132. Following treatment of HCT-116 cells with 20 μMMG-132 for 24 hours there was a readily apparent population of cellsthat had lost adhesion. While there was only a small increase in cleavedPARP, a product of caspase cleavage and activation, in the cells thatremained adherent, there was a large increase in cleaved PARP in thenon-adherent cells (FIG. 3C).

Pretreatment of adherent HT-29 cells for three days with variousconcentrations of BAY 11-7085 followed by a one-hour cell adhesion assaydemonstrated a significant and rapid inhibition of cell adhesion at highconcentrations of BAY 11-7085 (FIG. 3D), suggesting that NF-κB promotedcell adhesion. Even though BAY 11-7085 induced a loss of cell adhesion,it was important to show whether HT-29 cells were actually susceptibleto anoikis. When HT-29 cells were cultured in suspension for 24 hours ondishes coated with poly-HEMA, a substrate that completely inhibitedHT-29 cell adhesion, only a minority (17%) of the floating cells wereapoptotic by the annexin V assay. The fact that only a small fraction ofsuspended HT-29 cells were apoptotic, suggested that anoikis was not theprincipal mechanism responsible for the apoptotic activity of the NF-κBinhibitors.

d) Loss of Colon Cancer Adhesion Activates NF-κB

Various concentrations of BAY 11-7085 were added to HT-29 cells thatwere transiently suspended in trypsin-PBS for approximately 15 minutesbefore replating. The transiently suspended cells were quickly allowedto readhere in order to prevent anoikis. Alternatively, BAY 11-7085 wasadded to adherent HT-29 cell monolayers. BAY-11-7085 at allconcentrations tested caused a significant increase in apoptosis of thetransiently suspended HT-29 cells (FIG. 4A). On the other hand, even thehighest doses of BAY 11-7085 caused only a relatively small degree ofapoptosis of adherent compared with transiently suspended HT-29 cells(FIG. 5). Similar results were obtained with BAY 11-7082 at the samedoses.

Since a transient loss of cell adhesion greatly enhanced the apoptoticeffect of BAY 11-7085, we hypothesized that activation of NF-κB may beeffected by a loss of cell adhesion. To test this, DLD-1, HCT-116, andHT-29 cells were transiently suspended in trypsin-PBS and then quicklyallowed to readhere. Immunofluorescent studies on the localization ofthe p65 NF-κB subunit revealed nuclear p65 in cells one hour after beingtransiently in suspension (FIG. 4B). This was followed by a decrease innuclear p65 localization from 3-24 hours after replating. In fact, by 24hours, little nuclear p65 staining was seen although much cytoplasmicp65 staining was present. Similar results were obtained with DLD-1 (FIG.4C) and HCT-116 (FIG. 4D) colon cancer cell lines. These resultsdemonstrated that transient suspension of colon cancer cells resulted inactivation of NF-κB.

An NF-κB binding assay was used to demonstrate the activation of NF-κBby transient suspension. Adherent monolayers of DLD-1, HCT-116, andHT-29 cells were treated with increasing concentrations of BAY 11-7085for 8 hours were scraped off plates and subjected to an NF-κB activationassay. The scraped cells were lysed and added to 96-well platescontaining immobilized oligonucleotides corresponding to the NF-κBconsensus binding sequence (Trans-AM® assay). NF-κB binding to theimmobilized oligonucleotides was detected using a p65 monoclonal primaryantibody and a horseradish peroxidase-conjugated anti-mouse secondaryantibody. The assay showed that there was relatively little activationof NF-κB compared to the positive control (HeLa cells treated with TNFα)(FIG. 5A). However, when the transiently suspended cells were allowed toreadhere, a large induction of NF-κB activation occurred (FIG. 5A). Thusit was re-adhesion rather than just transient suspension that was thestimulus causing the large increase in NF-κB activation.

The same NF-κB binding assay was used to determine whether NF-κBinhibitors could diminish the activation of NF-κB by re-adhesion. HT-29cells transiently suspended with trypsin were mixed with increasingconcentrations of BAY 11-7085 then placed on culture dishes for 8 hours.Alternatively, adherent HT-29 cells were treated with increasingconcentrations of BAY 11-7085 for 8 hours. The cells were scraped, andall the cells were collected and lysed. The NF-κB binding assayconfirmed the strong activation of NF-κB caused by the readhesion oftransiently suspended HT-29 cells and demonstrated a dose-relatedinhibition of this NF-κB activation by BAY 11-7085 (FIG. 5B, transientlysuspended cells). On the other hand, there was a steady and low level ofconstitutive NF-κB activation in the adherent HT-29 cells that wasrelatively unaffected by treatment with increasing concentrations of BAY11-7085 (FIG. 5B, adherent cells).

Since trypsin can activate protease-activated receptor-2 in colon cancercell lines, which then results in increased cell proliferation, (Darmoulet al., 2001) NF-κB activation was determined in adherent and confluentDLD-1, HCT-116, Caco-2 and HT-29 cells that were removed from plasticdishes by trypsin or scraping. Scraping of the cells did not result inNF-κB activation (FIG. 5C). In addition, cells that were transientlysuspended in trypsin-PBS but not allowed to readhere, did not showsignificantly more activation of NF-κB by the TransAM assay (FIG. 5C).To determine whether the transient activation of NF-κB caused by cellreadhesion would render all of the colon cancer cells tested moresusceptible to BAY 11-7085-induced apoptosis, DLD-1, HCT-116, Caco-2 andHT-29 colon cancer cell lines were transiently suspended, treated withBAY 11-7085 and allowed to readhere. While DLD-1 and HT-29 cells showedthe same propensity for BAY 11-7085-induced apoptosis, Caco-2 cellsshowed a moderate increase, while HCT-116 cells did not (FIG. 5C).

The colon cancer cell lines used were chosen for differences in theirorigins. While DLD-1 and HT-29 cells carry mutated APC alleles, HCT-116cells do not, (Groden et al., 1995) however, HCT-116 cells carryactivating mutations in the β-catenin gene (CTNNB1), (Ilyas et al.,1997) whose gene product is normally regulated by the APC gene product.(Munemitsu et al., 1995; Rubinfeld et al., 1995) In addition, HCT-116cells do not express COX-2 while DLD-1 and HT-29 cells do. (Hsi et al.,2000; Shao et al., 2000; Tsuji et al., 1996) COX-2 is commonlyoverexpressed in colorectal tumors and plays roles in survival andmetastasis in colorectal cancers.

COX-2 overexpression plays a role in the susceptibility of the coloncancer cells to BAY 11-7085-induced apoptosis. 293 cells transfectedwith a ponasterone-inducible COX-2 construct (293-COX-2) were treatedwith BAY 11-7085. Treatment of 293-COX-2 cells with 1 μg/ml ofponasterone for 48 hours led to a large induction of COX-2 protein,while the uninduced cells showed no measurable COX-2 protein expressionby western blot (FIG. 5E) Uninduced 293-COX-2 cells showed no increasein apoptosis following treatment with BAY 11-7085 (FIG. 5F). However,ponasterone-induced 293-COX-2 cells demonstrated a marked increase inBAY 11-7085-induced apoptosis (FIG. 5E). This data indicated thatdifferences in COX-2 expression can influence the ability of BAY 11-7085to cause apoptosis.

Two other soluble NF-κB inhibitors, MG-132 and PDTC, were used to testtheir activity in the induction of apoptosis of colon cancer cellsduring readhesion. Compared with BAY 11-7085, MG-132 more potentlyinduced apoptosis of HT-29 colon cancer cells during readhesion. PDTCcaused apoptosis of HT-29 cells during readhesion as well.

The specific involvement of NF-κB in the apoptotic mechanism of BAY11-7085 was examined specifically. To accomplish this, DLD-1 and HT-29cells were transduced with an adenovirus containing the IκBsuper-repressor construct to specifically inhibit NF-κB. The IκBsuper-repressor was created previously by mutating two key serineresidues within the IκB gene resulting in an encoded IκB protein that isincapable of being targeted for ubiquitination and, hence, degradationby the proteasome. The transduced DLD-1 and HT-29 cells were thentransiently suspended and allowed to readhere at concentrations of BAY11-7085 below the in vitro apoptotic threshold (>20 μM) for these celllines. Expression of the IκB super-repressor in DLD-1 and HT-29 cellssignificantly lowered the apoptotic threshold of BAY 11-7085 comparedwith the controls. The effect of the IκB super-repressor was muchgreater for DLD-1 than HT-29 cells because the former expressed higherlevels of IκB super-repressor protein than the latter cells.Furthermore, the IκB super-repressor protein was functional in both celllines. Therefore, inhibition of NF-κB is important to the apoptoticeffect of BAY 11-7085 during colon cancer cell readhesion.

e) NF-κB Inhibitor Prevents Intraabdominal Metastasis In Vivo

Since BAY 11-7085 induced apoptosis of readherent HT-29 cells, an invivo model was used to test the ability of the drug to preventmetastasis by seeding of intraabdominal tissue with colon cancer cells.During colorectal cancer surgery, seeding of the peritoneal cavity bytumor cells with tumor cell implantation of the peritoneal surfaces canoccur. Seeding results from the transient suspension of cancer cells,through a loss of adhesion either naturally ro because of surgicaldisplacement, followed by readhesion to other tissues. The HT-29 andHCT-116 colon cancer cell lines were injected into the abdominalcavities of athymic mice that had been pretreated 24 hours earlier witheither intraperitoneal vehicle alone (DMSO) or BAY 11-7085 (1 mg/kg).The mice were then treated twice weekly with vehicle or BAY 11-7085 fora total of 21 days. Mice sacrificed 7 days after the introduction ofcolon cancer cells intraabdominally showed no evidence of tumoralimplantation of the parietal or visceral peritoneal surfaces.

After 21 days, there was clear evidence of metastases involving theparietal and visceral peritoneum of mice injected with HCT-116 cellsregardless of whether they had received vehicle or BAY 11-7085 (FIGS. 6Aand B). However, of the mice injected intraabdominally with HT-29 cellsand treated with BAY 11-7085, only 2 of 6 mice demonstrated parietalperitoneal metastases and none of the 6 showed any evidence of hepaticmetastases (Table 1). TABLE 1 Peritoneal and liver metastases followingintraperitoneal injections of HT-29 cells into athymic mice. TreatmentPeritoneal Metastases Liver Metastases DMSO 5/6 6/6 (n = 6) BAY 11-7085(n = 6) 2/6 0/6

Of the mice that had been injected intraabdominally with HT-29 cells andtreated with vehicle, 5 of 6 developed metastases of the parietalperitoneum and all 6 developed hepatic metastases (FIG. 6C and Table 1).Interestingly, the majority of the metastases involved ventral anddependent areas of parietal and visceral peritoneum, suggesting that thecolon cancer cells took some time to implant.

f) NF-κB Inhibitors Decrease Expression of Anti-Apoptotic Proteins

Inhibition of NF-κB increased the susceptibility of cancer cells toTNFα-induced apoptosis in other studies. (Han et al., 2000; Wang et al.,1999) Furthermore, TNFα is expressed by a number of colon cancer celllines including HT-29 cells. (Jung et al., 1995) In order to explore thepossibility that BAY 11-7082 and BAY 11-7085 may be sensitizing coloncancer cells to TNFα-induced apoptosis, HT-29 cells were pretreated witha monoclonal antibody, cA2, which inhibits TNFα binding to TNFαreceptors, (D'Haens et al., 1999) followed by treatment with BAY11-7085. A previous study demonstrated TNFα-induced activation of NF-κBin HT-29 cells. (Yamamoto et al., 1999) In order to ascertain whethercA2 alone had any measurable effects on colon cancer cells, DLD-1,HCT-116, and HT-29 colon cancer cells were treated with variousconcentrations of cA2 in a cell proliferation assay. Interestingly, cA2,at 10 and 50 μg/ml, caused a significant inhibition of colon cancer cellproliferation after several days of treatment (FIG. 7A). However,pretreatment of transiently suspended HT-29 cells with 50 μg/ml of cA2for 48 hours did not diminish BAY 11-7085-induced apoptosis of adherent(FIG. 7B). This indicated that endogenous TNFα was not necessary for BAY11-7085-induced apoptosis.

NF-κB regulates the expression of a number of genes, including c-IAP-1,c-IAP-2, TRAF-1, and TRAF-2, that encode proteins that mediate cellsurvival. (Stehlik et al., 1998; Wang et al., 1998; Wu et al., 1998) Nodecrease in c-IAP-1 protein expression occurred when transientlysuspended or adherent HT-29 cells were treated with 20 μM of BAY 11-7085for 8 hours (FIG. 7C). HT-29 cells did not express detectable levels ofc-IAP-2 by western blots. The same treatment of HT-29 cells did resultin a decrease in the protein expression of both TRAF-1 and TRAF-2proteins (FIG. 7C).

Recently, the anti-apoptotic protein, FLIP, which inhibits TNFα-mediatedapoptosis, was found to be regulated by NF-κB and overexpressed in avariety of cancers. (Elnemr et al., 2001; Irmler et al., 1997; Kreuz etal., 2001; Ryu et al., 2001; Tepper and Seldin, 1999) Interestingly,FLIP expression is upregulated by cell adhesion in endothelial cells andplays a role in inhibiting anoikis. (Aoudjit and Vuori, 2001) Disclosedherein FLIP was expressed in the DLD-1 and HT-29, but not the HCT-116,cell lines. Twenty μM BAY 11-7085 caused only a slight diminution in theexpression of the 54 kilodalton (long) isoform of FLIP in adherent, butnot transiently suspended, HT-29 cells (FIG. 7C). Although decreasedFLIP expression could be involved in BAY 11-7085-induced apoptosis ofHT-29 cells, it would not explain BAY 11-7085-induced apoptosis in DLD-1cells since they do not express FLIP (FIG. 7D).

Treatment of adherent colon cancer cells with BAY 11-7082, BAY 11-7085,or MG-132 inhibited colon cancer cell proliferation, tumorigenicity, andadhesion, and induced apoptosis. These effects were not uniform amongstthe selected colon cancer cell lines used. While the cell proliferationof all three cell lines were readily inhibited by BAY 11-7085, reducedtumorigenicity and apoptosis induction by treatment with BAY 11-7082 andBAY 11-7085 was only achieved in the DLD-1 and HT-29 cell lines. Resultsobtained using ponasterone-inducible COX-2 transgene expression in 293cells demonstrated a likely role for COX-2 protein expression orovexpression in the apoptotic response to BAY 11-7085.

While cells that became non-adherent following treatment with NF-κBinhibitors were apoptotic, those cells that remained adherent were not.In addition, pretreatment of HT-29 cells with BAY 11-7085 caused asignificant inhibition of cell adhesion at higher doses. Theproapoptotic effect of BAY 11-7085 was greater when it was added totransiently suspended HT-29 and DLD-1 cells (i.e.-just before plating)versus adherent cells. These results indicated that transient suspensionof colon cancer cells increased the susceptibility of the cells toapoptosis due to NF-κB inhibition.

When adherent DLD-1, HCT-116, and HT-29 cells were scraped off ofplastic dishes and assayed for NF-κB activation, the level of NF-κB DNAbinding was low relative to the positive control (HeLa cells stimulatedwith TNFα). When transiently suspended DLD-1, HCT-116, and HT-29 wereallowed to readhere, there was a rapid and large activation (similar tothe positive control) and increased nuclear localization of NF-κB. Thus,it was the readhesion of the transiently suspended colon cancer cellsthat activated NF-κB. This large activation of NF-78 B induced by thereadhesion of transiently suspended colon cancer cells was rapid andalmost totally inhibited by treatment with as little as 10 μM BAY11-7085. On the other hand, adherent colon cancer cells demonstrated alow but constitutive level of NF-κB activation, which could not beinhibited with doses of BAY 11-7085 up to 50 μM.

It appears that NF-κB inhibitors cause apoptosis of colon cancer cellsin a two-step process. First, the NF-κB inhibitors inhibit cell adhesionof adherent colon cancer cells in vitro. The re-adhesion of thesefloating cells causes a large and transient activation of NF-κB. Thisrenders the readherent cells exquisitely susceptible to NF-κBinhibitor-induced apoptosis. The fact that the vast majority oftransiently suspended HT-29 and DLD-1 cells, which were allowed toreadhere, became apoptotic following treatment with BAY 11-7082 or BAY11-7085, indicated that NF-κB is an important survival factor forcertain cancer cells, such as colon cancer cells, during the process ofre-adhesion, particularly cancer cells that express COX-2 and/or mutantAPC genes.

As metastatic cancers must transiently suspend to pass through thecirculatory system and then readhere to be invasive, these resultsindicate that the compositions disclosed herein can be used for theprevention of metastasis The process of metastasis has been proposed toinvolve a number of sequential steps: invasion, dissociation,intavasation into the circulatory or lymphatic systems, dissemination,arrest in the microcirculation, extravasation, and invasion of distanttissues. (Engers and Gabbert, 2000) Each of these steps involves dynamicchanges in cell adhesion including a complete absence of adhesion duringdissemination. Although circulating cancer cells in humans can beabundantly found in venous blood samples of advanced cancer patients,(Mehes et al., 2001) Fortunately, the process of metastasis issurprisingly inefficient with as few as 0.05% of circulating tumor cellsproducing stable metastases. (Liotta et al., 1974; Nicolson, 1991;Weiss, 1985) Although many circulating tumor cells become arrested inthe microcirculation, the majority of these cells remain viable andcapable of extravasation in vivo. (Chambers et al., 1995) Thus, the ratelimiting step in metastasis is the colonization of distant tissues byextravasated tumor cells (Chambers et al., 1995).

While the five-year survival rate for early stage colorectal cancers(TNM I-II) without metastases is greater than 80%, for late-stagedcancers (TNM III-IV) with metastases it is less than 50%.(Boland, 1999)Most newly diagnosed colorectal cancers are TNM stage III-IV, whichmeans that most colorectal cancer patients already have metastasis ofthe primary tumor by the time of presentation. In fact, micrometastasisto lymph nodes was detected by RT-PCR of carcinoembryonic antigen in 54%of patients deemed to be TNM stage II by current staging methods.(Liefers et al., 1998). In this study, the five-year survival of thepatients with lymph node micrometases was 50% versus 91% for thosewithout micrometases. This means that the stage II patients withmicrometastases behaved clinically more like TMN stage III patients withregards to survival. Thus, a greater proportion of newly diagnosedcolorectal cancers already have metastases than previously thought.Indeed, breast cancer cells in the blood of patients with advancedbreast cancer were found as frequently as 1 per 1000 mononuclear cells.(Mehes et al., 2001) Although the majority of these cells wereapoptotic, a minority were not, demonstrating that a significant numberof cancer cells invade into the circulatory system and survive.

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Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art. The references disclosed are alsoindividually and specifically incorporated by reference herein for thematerial contained in them that is discussed in the sentence in whichthe reference is relied upon.

1. A method for inhibiting cancer cell proliferation comprisingadministering a NF-κB inhibitor to a subject, wherein the NF-κBinhibitor causes an NFkB inhibition, wherein the subject has cancercells which are proliferating, wherein the cancer cells are not myelomacells.
 2. A method of promoting cancer cell apoptosis comprisingadministering a NF-κB inhibitor to a subject, wherein the NF-κBinhibitor causes an NF-κB inhibition, wherein the subject has cancercells, wherein the cancer cells are not myeloma.
 3. A method ofinhibiting readhesion of cancer cells to a surface comprisingadministering a NF-κB inhibitor to a subject, wherein the NF-κBinhibitor causes an NF-κB inhibition, wherein the subject has cancercells.
 4. A method of inhibiting metastasis of cancer cells comprisingadministering a NF-κB inhibitor to a subject, wherein the NF-κBinhibitor causes an NF-κB inhibition, wherein the subject has cancercells.
 5. The method of claim 4, wherein the NF-κB inhibtor inhibitsintraabdominal metastasis.
 6. The method of claim 4, wherein the NF-κBinhibitor inhibits hepatic, parietal or peritoneal metastasis.
 7. Amethod of inhibiting tumorigenesis comprising administering a NF-κBinhibitor to a subject, wherein the NF-κB inhibitor causes an NF-κBinhibition, wherein the subject has cancer cells.
 8. The method of claim1, wherein the cancer is an abdominal cancer, hepatic cancer, peritonealcancer, parietal cancer, rectal cancer, stomach cancer, or colon cancer.9. The method of claim 1, wherein the cancer cells utilize NF-κB formitogenesis.
 10. The method of claim 1, wherein the cancer cells utilizeNF-κB for readhesion
 11. The method of claim 1, wherein the cancer cellcomprises an APC mutation.
 12. The method of claim 1, wherein the cancercell does not contain an activating mutation on β-catenin.
 13. Themethod of claim 1, wherein the cancer cell expresses the COX2 gene. 14.The method of claim 12, wherein the cancer cell overexpresses the COX2gene.
 15. The method of claim 1, wherein the cancer cell does notexpress the COX2 gene.
 16. The method of claim 1, wherein the cancercell is related to a cancer cell line.
 17. The method of claim 14,wherein the cancer cell line is a DLD-1 cell line or a HT-29 cell line.18. The method of claim 1, wherein the cancer cells are colon cancercells.
 19. The method of claim 1, wherein the cancer cells are rectalcancer cells.
 20. The method of claim 1, wherein the cancer cells arenot adenocarcinoma cells.
 21. The method of claim 1, wherein inhibitingcancer cell proliferation is independent of TNFα activated apoptosis.22. The method of claim 2, wherein promoting cancer cell apoptosis isindependent of TNFα activated apoptosis.
 23. The method of claim 3,wherein inhibiting readhesion of cancer cells to a surface isindependent of TNFα activated apoptosis.
 24. The method of claim 4,wherein inhibiting metastasis of cancer cells is independent of TNFαactivated apoptosis.
 25. The method of claim 7, wherein inhibitingtumorigenesis is independent of TNFα activated apoptosis.
 26. The methodof claim 1, wherein the NF-κB inhibitor causes a decrease in theexpression of anti-apoptotic proteins.
 27. The method of claim 1,wherein the NF-κB inhibitor inhibits IκB phosphorylation.
 28. The methodof claim 1, wherein the NF-κB inhibitor inhibits TNFα induced NF-κBactivation.
 29. The method of claim 1, wherein the NF-κB inhibitor is anolefin.
 30. The method of claim 1, wherein the NF-κB inhibitor is anolefin having at least oneelectron-withdrawing group.
 31. The method ofclaim 1, wherein the NF-κB inhibitor is an olefin having at least twoelectron-withdrawing groups.
 32. The method of claim 29, wherein theelectron-withdrawing group comprises a cyano group, a sulfo-oxy group, aphospho-oxy group, a carboxyl group, a nitro group, a halogen, ahalogenated alkyl group, an unsubstituted aromatic ring, or asubstituted aromatic ring having at least one cyano group, sulfo-oxygroup, phospho-oxy group, carboxyl group, hydroxyl group, amino group,ether group, halogenated alkyl group, halogen, or nitro group.
 33. Themethod of claim 31, wherein the phospho-oxy group has the structure

wherein R¹ is hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl,aralkyl, or substituted or unsubsituted aromatic. [N&R will define eachof these terms in the specification.]
 34. The method of claim 31,wherein the sulfo-oxy group has the structure

wherein R² is hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl,aralkyl, or substituted or unsubsituted aromatic.
 35. The method ofclaim 1, wherein the NF-κB inhibitor is an olefin having a cyano groupand a sulfo-oxy group having the structure

wherein R² is hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl,aralkyl, or substituted or unsubsituted aromatic.
 36. The method ofclaim 1 wherein the NF-κB inhibitor has the structure

wherein R², R³ and R⁴ are, independently, hydrogen, alkyl, halogenatedalkyl, alkenyl, alkynyl, aralkyl, or substituted or unsubsitutedaromatic, wherein the compound is the E- or Z-isomer.
 37. The method ofclaim 35, wherein R³ and R⁴ are hydrogen.
 38. The method of claim 35,wherein R² is methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tertiary butyl, substituted or unsubstituted phenyl, or benzyl.
 39. Themethod of claim 35, wherein R² is a phenyl group having at least onealkyl group.
 40. The method of claim 35, wherein the compound is theE-isomer.
 41. The method of claim 1, wherein the NF-κB inhibitor has thestructure


42. The method of claim 1, wherein the NF-κB inhibitor has the structure


43. The method of claim 1, wherein the NF-κB inhibitor has the structure

wherein R₆ and R₇ are, independently, hydrogen, alkyl, alkenyl, alkynyl,aralkyl, or substituted or unsubstituted aromatic, or R₆ and R₇ togetherform a ring with the nitrogen atom, X and Y are, independently, oxygenor sulfur, or the pharmaceutically-acceptable salt, ester, or amidethereof.
 44. The method of claim 43, wherein X and Y are sulfur, and R₆and R₇ is (CH₂)₄.
 45. The method of claim 1, wherein the NF-κB inhibitorcomprises at least one amino acid residue.
 46. The method of claim 1,wherein the NF-κB inhibitor has at least one leucine residue.
 47. Themethod of claim 1, wherein the NF-κB inhibitor comprises three leucineresidues.
 48. The method of claim 1, wherein the NF-κB inhibitor isN-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide.49. The method of claim 40, wherein the NF-κB inhibitor is BAY-11-7082.50. The method of claim 40, wherein the NF-κB inhibitor is BAY-11-7085.51. The method of claim 1, wherein the NF-κB inhibitor is MG-132. 52.The method of claim 1, wherein the NF-κB inhibitor is PDTC.
 53. Themethod of claim 1, wherein the NF-κB inhibitor directly inhibits NF-κB.54. The method of claim 1, wherein the NF-κB inhibitor indirectlyinhibits NF-κB.
 55. The method of 52, wherein the NF-κB inhibitorinhibits expression of NF-κB.
 56. The method of 52, wherein the NF-κBinhibitor inhibits translation of NF-κB.
 57. The method of claim 1,wherein the NF-κB inhibitor inhibits NF-κB transport into the nucleus.58. A method of inhibiting cancer cell proliferation in a subject,comprising testing for an adenomatous polyposis coli (APC) genemutation, and if the mutation is detected, administering an effectiveamount of an NF-κB inhibitor to the subject.
 59. The method of claim 58,wherein the NF-κB inhibitor comprises BAY 11-7085.
 60. The method ofclaim 58, wherein the NF-κB inhibitor comprises BAY 11-7082.
 61. Amethod of inhibiting cancer cell proliferation in a subject comprisingtesting the subject for COX2 expression, and if there was COX 2expression, administering an NF-κB inhibitor to the subject.
 62. Themethod of claim 61, wherein the NF-κB inhibitor comprises BAY 11-7085.63. The method of claim 61, wherein the NF-κB inhibitor comprises BAY11-7082.
 64. A method of inhibiting cancer cell proliferation in asubject comprising administering an NF-κB inhibitor to the subject,wherein the subject has had a tumor resected.
 65. The method of claim64, wherein the NF-κB inhibitor is administered prior to the resection.66. The method of claim 64, wherein the NF-κB inhibitor is administeredprior to the resection.
 67. The method of claim 64, wherein the NF-κBinhibitor is administered within 10 days of the resection.
 68. Themethod of claim 64, wherein the NF-κB inhibitor is administered within 5days of the resection.
 69. The method of claim 64, wherein the NF-κBinhibitor is administered within 1 days of the resection.
 70. The methodof claim 64, wherein the NF-κB inhibitor is administered within 10 hoursof the resection.
 71. The method of claim 64, wherein the NF-κBinhibitor is administered within 1 hour of the resection.
 72. The methodof claim 64, wherein the NF-κB inhibitor is administered within 0.5hours of the resection.